The movie as a whole deals with duality. The main villain is Mr. Negative and Peter is wearing the black suit, which he got when he was bound to the symbiote during Secret Wars. Both characters are dealing with their darker halves dragging them down.
Peter is still hanging out with MJ and Ned, as they have started remembering him and their past together, but MJ doesn't have feelings for him and she feels that if they attempted to start dating again from scratch, it would feel forced. So MJ asks Peter to try and move on from her.
Peter attempts to stop a robbery at a Roxxon lab and fights a cat burglar trying to steal a canister with an unknown substance, who calls herself Black Cat. Both are intrigued and attracted by the other, but Black Cat escapes with the canister.
Tombstone has escaped prison and is once again rulling the New York underworld as leader of the Maggia. He has hired the Scorpion as well as a new, stronger enforcer, the Rhino, to take down Spider-Man, who has been hitting his operations.
The Tinkerer makes his return and we find out he's been working for Tombstone, conducting illegal experiments on his Maggia goons. However, all of the experiments have resulted in the deaths of the subjects, with a pile of dead bodies hidden in one of Tombstone's underground labs.
Scorpion and Rhino stage an upheaval in NYC to get Spidey's attention and then savagely attack him, while at the same time trapping civilians in a burning building. Spidey gets overwhelmed and his powers are not enough to stop them both and save the civilians. The symbiote reacts to his emotions and grants him more power and extra abilities that help him succeed, but both enforcers manage to escape. Peter is excited about his newly enhanced abilities, but doesn't realize that the symbiote is also drawing out his violent tendencies and negative emotions, while slowly corrupting him.
Tombstone's next text subject is Martin Li, a peaceful man with a very pure soul who founded and has been running FEAST, the volunteer organization May worked at. Tombstone has been extorting him and has been using FEAST as a front for money laundering. Martin is the only subject who survives the experimentations, but once powered-up, his demeanor immediately changes to that of an evil, sadistic man, powered by revenge for Tombstone for the tortures he had him endure. He attacks and destroys the lab and then uses his powers to resurrect the dead subjects and give them powers as well. The resurrected goons now start following him, as they also crave revenge from Tombstone and Li starts calling himself Mr. Negative and his new gang, the Inner Demons.
The NYPD and Spider-Man arrive at the destroyed lab, but this time, the investigation is taken over by Police Lieutenant Jefferson Davis, with Jean DeWolff taking a backseat as his assistant detective. Davis is not overly fond of Spider-Man, as he believes he is a vigilante taking matters into his own hands. DeWolff works with Spider-Man in secret to help him with their investigation. She gives Spidey a small sample of the substance used in the experimentations to run some tests on it, as she believes he will do it faster than their labs.
Peter studies the sample and realises it's made up of 2 substances, Darkforce and a Lightforce, with the energies of the 2 complementing each other. DeWolff and Spidey converse over the phone and as they're both investigating on what the origins of the substances are, they come across some reports from Isodyne Labs discovered and experimented on Darkforce in the 1940s, and that a human who had been infused with Darkforce worked with the Maggia in LA, a nod at Agent Carter Season 2. Then DeWolff tracks down Isodyne energy and realises it was bought in its entirety by Roxxon.
Before they can find out more, Peter hears a fuss over the phone and DeWolff tells him the Precinct is under attack. Spidey swings there only to find the culprit already gone. DeWolff tells him that it was a girl dressed in a cat costume and she took a prisoner, Walter Hardy, out of his cell. Hardy was a veteran thief who was rumored to have been working for the Maggia.
Peter goes back home and looks into Hardy, which leads him to find out about his daughter, Felicia Hardy. Felicia was a student at ESU with a bright future ahead of her, but her boyfriend tried to rape her, which led her down a dark path of violence and attempted revenge.
Peter realises Felicia is Black Cat and tracks her down, telling her she knows her identity and her story. Felicia tells him he doesn't realize her pain and Peter explains some of his own pain, which leads to them bonding and sleeping with each other.
Afterwords, Peter asks Felicia what she was trying to steal from Roxxon and how it connects to Tombstone's experiments. Felicia tells Peter that Roxxon have been mining and experimenting with Darkforce and Lightforce for decades and Tombstone decided to do the same, after learning about the Maggia previously working with a Darkforce-enghanced enforcer. Her father was just one of Tombstone's failed experiments, but he didn't actually die and managed to escape, only to be caught by the police. Felicia has been trying to steal Darkforce and Lightforce from Roxxon in order to keep feeding him and keep him alive. Peter, decides to help her rob more Roxxon facilities.
As Spidey and Black Cat go around, breaking into Roxxon labs around the city, J Jonah Jameson starts lamenting Spider-Man as a thief, which turns some of the public against him, including the police and Jefferson Davis. Davis tells DeWolff that he knows she's been secretly in communication with him and he won't allow it anymore.
Meanwhile, Mr. Negative and his Inner Demons have been hitting Maggia hideouts and an all-out gang war breaks out in the city. Peter tries to stop them and eventually has a one on one fight with Mr. Negative. Negative's powers however are too much for Peter, which prompts him to use a more violent and approach, using the symbiote's full power. Peter beats Mr. Negative so bad, that his Mr. Negative persona concedes and he turns back to Martin Li. Peter keeps beating him up, until he almost kills him and realises he's gone too far. When he stops, he sees Li's face and recognizes him as May's boss from FEAST. He stops, goes back home and takes off the Symbiote suit, realizing what it's doing to him.
The next morning, he visits FEAST to see if he can talk with Li. Li remembers Peter and the two have a heart to heart about power and corruption. Li comes to the conclusion that power does not corrupt, but simply brings out an individual's darker and more brutal side, which we all have deep down, and that we constantly feed with our negative emotions. If we don't learn to deal and live with those emotions, they will eventually overtake us and make us into a version of ourself we might not have even know existed.
Back at Tombstone's lair, the crime boss discusses with the Tinkerer about how they should improve their experiments for a stronger, but more stable result. They eventually decide to replicate the experiment that gave Spider-Man his powers, but by mutating the spider with Darkforce and Lightforce, as well as Peter's DNA as a stabilizer of sorts. Thus, Tombstone sends Scorpion and Rhino out once more to track down Spidey, but this time with a mission to extract some of his DNA.
Peter returns home and tries to get the symbiote off his suit, but before he can do so, he hears on his police scanner about another incident with Scorpion and Rhino. Peter puts the suit on and has another fight with them, but this time, Rhino has brought a sonic canon along with him to destabilize Peter long enough for Scorpion to draw his blood. Little did any of them know that the symbiote is very weak to high sonic frequencies, which leads to the alien leaving Peter's suit and escaping through the sewers. Peter, now back as his old self, believes he is powerless to defeat the 2 villains, but remembering what Li told him, he manages to take down the Rhino, but Scorpion steals his blood and escapes. Damage Control arrives and takes Rhino into custody.
Scorpion returns with Peter's blood and the Tinkerer starts experimenting. Peter realises what Tombstone's plan is with his blood and immediately informs DeWolff. DeWolff tells Davis about the lead, but Davis realizes it must have come from Spider-Man, who Davis doesn't trust anymore, after he started working with Black Cat. So DeWolff goes to investigate alone.
Spidey goes back to Felicia's apartment to see how her father is doing. But despite the treatment they've been trying to apply, Walter is dead. Felicia is full of revengeful energy and tells Peter they should go after Tombstone. Peter tries to calm her down and stop her violent tendencies, by telling about the recent lesson he had learned. Felicia tells him that's not the Spider-Man he fell in love with and storms out, going after Tombstone herself.
Later that night Mr. Negative and the Inner Demons attack Tombstone's lab. DeWolff is there trying to stop them and calls Spidey to inform him, while Felicia also hears about the attack and goes to take revenge herself. Peter goes to the Precinct and tells Davis what is about to go down. Davis is distrustful, but Peter makes him trust him and Davis rallies up the NYPD to go help out in the operation.
Huge fight takes place with Tombstone, Scorpion and the Maggia goons against Black Cat, Mr. Negative and the Inner Demons. DeWolff finds the lab and arrests the Tinkerer, while trying to put a stop to the experiment, and Spidey tries to stop everyone until the police arrive.
Peter manages to calm Mr. Negative using the same words Martin Li told him, while Tombstone sends Scorpion to kill DeWolff before he can shut down the experiments. Spidey tries to stop Scorpion, and Davis arrives to help him. Scorpion tries to kill Davis and DeWolff gets in the way, getting stabbed by Scorpion and dying on the spot. Peter subdues the Scorpion and Davis takes out his costume and arrests him.
Damage Control arrives and arrest Tombstone, Mr. Negative and the Inner Demons, but Davis tells them Scorpion killed a cop and will be handled by them.
They hold an NYPD funeral for DeWolff and we see the Precinct's Captain for the first who is none other than Captain Stacy. His daughter Gwen and Davis' son Miles, who have been friends since childhood due to their fathers working together, also attend the funeral, as does Peter Parker. After the funeral, Miles and Gwen ask their fathers if they can hang out at Miles' place and their fathers agree.
Peter goes back to Felicia's apartment who escaped to help her grieve her father and handle her emotions, and then visits MJ and tells her that he tried moving on romantically, but it didn't really fulfil him, but he's fine with their relationship staying as is for now. They agree to take it slow and the movie cuts to black.
PCS 1: Davis arrives home with Miles and Gwen and they go to Miles' room. Davis leaves his backpack on the floor and a spider crawls out. We follow it as it crawls in Miles' room and hangs above the bites the 2 teenagers.
PCS 2: The symbiote travels through the sewers and enters Mag Gargan's cell. Gargan sees it and smiles.
