The game begins with a tutorial level where Spider-Man stops the H-Bombers from blowing up the Carlyle building. The next day, Spider-Man investigates several humanoid lizards seen in Central Park, and continues his battle against the H-Bombers, stopping them from blowing up the Daily Bugle's Printing Plant and Regional Office. That night, while Peter and Mary Jane are discussing their problems in Central Park, an alien symbiote inside a meteor crashes nearby and attaches itself to Peter's shoe. After taking Mary Jane home, Peter is attacked by Harry, who became the "New Goblin" to avenge his father's death. Peter defeats Harry, knocking him out in the process, and takes him to the hospital to recover. He then returns home, where he is enveloped by the symbiote, creating a black Spider-Man suit that enhances his powers and increases his aggressiveness. Elsewhere, an escaped convict named Flint Marko becomes the Sandman after accidentally falling into an experimental particle accelerator that fuses his body with the surrounding sand.
The following day, the H-Bombers attack the Daily Bugle and kidnap J. Jonah Jameson, but Spider-Man manages to rescue the latter. He then fights and defeats the Bombers' leader, who is revealed to be businessman Luke Carlyle, who turned to crime after his company was ruined by a story published by Jameson. Later, Peter investigates the disappearance of his science teacher, Dr. Curt Connors, and discovers that he has been conducting experiments on himself with a lizard DNA serum, one of which transformed him into the Lizard. Chasing the Lizard through the sewers, Spider-Man discovers that he has transformed numerous other people into creatures like himself, intending to have them invade New York. Although Spider-Man manages to foil his plan, the Lizard escapes further into the sewers. When Spider-Man finds him again, the Lizard is fighting Kraven the Hunter, who has been trying to hunt him. Spider-Man intervenes in the confrontation and defeats Kraven, before overpowering the Lizard and restoring Connors to his human form.
After an assignment from Jameson leads Spider-Man to encounter a vampire, he defeats the creature and brings it to Connors, who recognizes the vampire as renowned biochemist Dr. Michael Morbius. With Morbius claiming that his wife Shriek was responsible for his transformation, Spider-Man tracks her down and defeats her, but she escapes. After battling Shriek's gang, the Waste Tribe, Spider-Man discovers her whereabouts and brings Morbius to her so that she could cure him. However, she instead turns Morbius against Spider-Man, who defeats him. Shriek then tries to fight Spider-Man herself, but he is able to counter her powers using the symbiote. Defeated, Shriek uses her powers to cure Morbius before falling into a coma. Spider-Man leaves the unconscious Shriek in Morbius' and Connors' care and departs.
Later, Peter discovers that Eddie Brock has been masquerading as a black suited Spider-Man so that he could 'expose' Spider-Man as a criminal and earn a promotion. After humiliating Brock as Spider-Man, Peter exposes the scheme and earns the promotion in his place, causing Brock to swear revenge against both Spider-Man and Peter. Shortly afterwards, Spider-Man spots Marko robbing an armored van and chases him into the subway, where they fight. Discovering that water is Marko's weakness, Spider-Man bursts open a pipe, releasing water that reduces Marko to mud and washes him away. Later that night, after becoming violent with Mary Jane during a date, Peter realizes that the symbitoe is negatively influencing his behavior, and attempts to remove it at a nearby church. He is successful thanks to the church bell's sonic vibrations, which weaken the symbiote, but the creature then bonds with Brock, who followed Spider-Man to the church. As Venom, Brock, having deduced that Peter is Spider-Man, finds a still-living Marko and blackmails him into helping him kill Spider-Man, threatening to murder his daughter. The pair kidnap Mary Jane to lure out Spider-Man, while a recovered Harry comes to his friends' aid. Harry rescues Mary Jane and Marko's daughter and helps Spider-Man defeat Marko before Venom kills him. Spider-Man ultimately defeats Venom after using sonic vibrations to weaken him, but Brock dies after the symbiote leaves his body.
Afterwards, Spider-Man reconciles with Mary Jane, while Marko is reunited with his daughter. He apologizes to Spider-Man and leaves peacefully. The game ends with Spider-Man resuming his never-ending battle against crime, while stating that the only way to honor and remember those he loves is by never giving up being Spider-Man.