Doctor Octopus has kidnapped Aunt May and re-assembled the Sinister Six supervillains; Mysterio, Sandman, Vulture, Scorpion, and Kraven. The player controls Spider-Man through six side-scrolling levels, battling various minor thugs and minions until defeating the supervillain at the end of each level.

Spider-Man can jump, punch, kick, collect web cartridge icons to shoot web projectiles, swing on web lines, and climb up certain walls.

The level design features two stages before a third stage, of which is a boss fight. Kraven the Hunter's battle is the only boss fight which features a short cutscene before the fight, but after the first two stages.

Dr. Otto Octavius (Doctor Octopus) holds a meeting of the Sinister Six during the main titles of the game. He tells them to hunt down Peter Parker, Spider-Man's photographer, to reach their hated enemy. Upon reaching the Parker household, Sandman and the Scorpion realize that Peter isn't there. They decide to take May Parker (Aunt May) instead, and leave a message for Peter to tell Spidey to go to the Coney Island Pier. In the first level, the player must get past a carnival and through a sewer to get to the Mysterio boss fight, where Mysterio uses look-alike illusions to try to kill Spider-Man. After his defeat, Mysterio questions Spider-Man about heading to the pier. In the second level, the player must get past a dock and retrieve a key (found at the end of the second part of the level, held by an enemy) to unlock a gate in the pier and fight the Sandman. The Sandman, upon defeat, mumbles something about the Vulture at the World Trade Center before passing out. In the third level, the player must ascend the World Trade Center to fight the Vulture at the top while opening doors to get through to the roof. After Vulture's defeat, he drops a paper from Madison Square Gardens. In the fourth level, the player must go through Madison Square Gardens in order to reach the basketball court, where they must fight the deadly Scorpia, telling them to go to Central Park. In the 5th Central Park level, the player must progress through the park to reach Kraven the Hunter, where he admits the kidnapping lacks honor, and to go to the Empire State University. In the 6th and final level, the player must progress through the ESU campus to reach Dr. Octopus and rescue Aunt May. Peter thinks about getting Aunt May an unlisted address.

Minigames

A secret minigame can be unlocked at the game's title screen. This is a variation on the Game & Watch Gallery game, Fire. A huge building is burning, and teddy bears are continually leaping out of a window. Spider-Man and Dr. Octopus must move a stretcher around to keep the teddy bear from falling and to bounce it into an ambulance.