At the start of the game, Spider-Man finds a group of thugs attacking a woman. He fights them off, only to have the woman he saved call him a freak and run away. Spider-Man is then seen chasing Sandman through New York City. They engage in combat and Sandman is defeated, just before Rhino throws a car at Spider-Man, who avoids it. Rhino manages to escape and then the Triskelion can be seen in flames. It is later revealed that the fire was caused by an explosion, which let all super-villains free from the prison, along with symbiotes created from a mixture of the venom suit and OZ formula. Throughout the game, Spider-Man has to fight several villains which appear in Ultimate Spider-Man comics, such as Electro, Green Goblin and Rhino. Many of the villains' followers have to be fought as well, like symbiotes, thugs and goblins.
After fighting Electro, the police talks to Spider-Man, saying that if he keeps fighting villains, they would have to send Roxxon security robots to get him, as they have the situation under control. After fighting Venom twice and facing a symbiote army, Spider-Man is informed by S.H.I.E.L.D. agents that Norman Osborne (Green Goblin) has captured some baseball players, as well as the mayor and threatened to transform them into goblins unless the whole city is given to him. Spider-Man fights Doctor Octopus and then heads to the stadium, where he finds Green Goblin, who escapes. After chasing Norman through Manhattan, Spider-Man fights and defeats him. After defeated Green Goblin is taken by S.H.I.E.L.D. agents, who thank Spider-Man for his help and informs him that Dr. Connors (Lizard) had been working to create an antidote for the symbiotes left in the city.
At the end of the game, Spider-Man is sitting on a bench, when the woman which appears in the beginning of the game, comes to him and says he is not a freak after all, before photographing the two of them with her phone. Spider-Man tells her that he would appreciate if she did not upload the photograph to the internet, and then goes away.