Peter Parker and his college science professor Dr. Curt Conners explore neogenics as a method of limb regeneration. But while experimenting on himself, Connors accidentally becomes the Lizard, a half-man, half-reptile monstrosity that terrorizes Manhattan and subsequently kidnaps his own wife. Spider-Man must stop the Lizard, but his task is doubly difficult because he doesn't want to hurt the good friend he knows is trapped inside.

Norman Osborn hires Spencer Smythe to design a machine capable of destroying Spider-Man—the Black Widow Spider Slayer. But when Flash Thompson puts on a Spider-Man costume as a party gag, the deadly machine spots him, and he's carried off in Peter's place.

With Spencer Smythe dead, his son Alistair turns to the Kingpin to help seek his revenge on Spider-Man and others he deems responsible for his father's death. He captures Eddie Brock, J. Jonah Jameson, Norman Osborn, and Flash Thompson. He then turns his sights on Spider-Man and sends three slayers after him. Spider-Man only manages to hold his ground while fighting them individually. However, he is not so lucky when they combine into a super slayer and carry him off to Smythe.

Dr. Otto Octavius becomes the mad Dr. Octopus when his robotic arms become fused to him in a laboratory accident. He then seeks revenge for the humiliation the Hardy Foundation has caused him, so he kidnaps Felicia Hardy and holds her ransom.

The Master of Illusion, Mysterio has fooled the city and J. Jonah Jameson into thinking Spider-Man has turned to crime. However, Spider-Man manages to find Mysterio, only to be brought to a trap of special effects.

J. Jonah Jameson hires Dr. Farley Stillwell, a genius in genetic engineering, to make a new superhero that can match and defeat Spider-Man. With a willing participant, Mac Gargain, they engineer the DNA of a scorpion and him together to form a new hero, the Scorpion. However, when Gargain thinks they have turned him into a freak, the hero turns into a monster and goes on a rampage. Spider-Man is forced to step in to stop the creation from killing its creator.

Robbie has set Peter up with a job interview with a researcher by the name of Dr. Mariah Crawford. However, when he gets to her lab, he finds her under attack by a monstrous man who deems himself Kraven the Hunter. Spider-Man leads Crawford away from Kraven to the roofs. There, Crawford tells Spider-Man that Kraven was not always that way, that in fact she loved him as Sergei Kravenoff. Crawford tells of an accident years back that would have killed Sergei had she not used a dangerous potion that saved his life and gave him the strength and instinct of an animal, but turned him mad. Meanwhile, while looking for Crawford at her apartment, Kraven learns of her connection to Robbie and kidnaps him. He then threatens to kill Robbie if Spider-Man does not come to him with Crawford.

Spider-Man goes after Eddie Brock for libeling him, as he is now being stalked by bounty hunters at every turn. After being reassured by Mary Jane that he has changed, he seeks help from Dr. Curt Connors who discovers that the suit is alive, and Peter is the host organism.

The Kingpin is furious at Spider-Man and sends the Shocker and the Rhino out to finish him off with the aid of another new invention of Alistair Smythe. Meanwhile, Eddie Brock, as the symbiote creature Venom, is busy playing cat and mouse with Spider-Man, intruding on Peter's private life and making it clear he knows the web-slinger's secret identity. To make matters worse, Peter soon realizes that the pumped-up Eddie as Venom is stronger than he is.

Norman Osborn hires the Hobgoblin, a hitman and thief, to take out Osborn's "silent" partner Wilson Fisk, the Kingpin. When he fails in the attempt, the Hobgoblin changes sides, kidnapping Norman's son, Harry. Harry just happens to be best friends with Peter Parker.

When Aunt May is slightly injured by one of the Hobgoblin's pumpkin bombs during the kidnapping of Harry, Spider-Man enters the fray with a vengeance. After several battles, the trail leads Peter to Oscorp and Harry's own father. For Peter, rescuing his friend is easy compared to explaining to Harry that his own father is involved.

An international spy and hitman known as the Chameleon plans to assassinate two major diplomats on the brink of signing the peace proposal of the century. S.H.I.E.L.D. director Nick Fury solicits J. Jonah Jameson's assistance in an effort to prevent the "human shape-shifter" from derailing the proposal signing. But only Spider-Man with his super keen "spider-sense" can discern who is really whom and stop the criminal before it's too late.