Spider-Man must travel through several different locations in New York City in an effort to save his wife, Mary Jane, who had been kidnapped by Venom. Along the way, he fights old nemeses who taunt Spider-Man as to the whereabouts of his wife.
- Spider-Man: main character - Peter Parker, bitten by a radioactive spider, now fights crime as the Amazing Spider-Man. He begins the game through a phone call with Mysterio, who tells him that his wife, Mary Jane, has been kidnapped. Spider-Man travels through the city, fighting criminals and arch-villains in an attempt to get Mary Jane back.
- Mary Jane Watson-Parker: Also known as MJ, Mary Jane is the wife of Peter Parker. She has been kidnapped by Spider-Man's worst enemies.
- Mysterio - first boss - This former special effects wizard appears to know Spider-Man's true identity, and uses that knowledge to trap him. Mysterio is the first boss and is found at the end of an alleyway. He uses his smoke effects to try and confuse Spider-Man.
- Hobgoblin - second boss - The heir to the Green Goblin, the Hobgoblin awaits Spider-Man at the top of a building. He drops bombs on Spider-Man while flying through the air on his goblin glider.
- Scorpion - third boss - He taunts Spider-Man into entering the subway, where he uses his prehensile tail to try and impale Spider-Man.
- Rhino - fourth boss - Through Central Park and at the dead end in an alleyway, the Rhino uses his massive weight to try and run Spider-Man through. He is of sub-par intelligence. NOTE: In the game, this boss is actually listed as Neanderthal, not Rhino. It is still not clear whether this boss is actually Rhino or not.
- Doctor Octopus - fifth boss - A brilliant doctor before an accident fused mechanical arms to his body, Doctor Otto Octavius uses his new appendages to try and stop Spider-Man at the end of the rooftop of the second building.
- Venom - sixth and final boss - The true abductor of Mary Jane, Eddie Brock has had it out for Peter Parker even before he bonded with an alien symbiote. He has powers similar to Spider-Man, using webbing to his advantage during their sewer battle.
Spider-Man has three extra lives, and then three continues. However, there are no passwords or secret codes. The intermissions scenes between each level feature Spider-Man trading catchy barbs with a supervillain on a cell phone or a walkie talkie in order to find out where he should go next.
- Jump - Spider-Man can jump a normal and a double height. When he jumps double, he can swing on a web.
- Punch - Spider-Man's primary standing attack is a solid punch to the jaw.
- Low Kick - While crouching, Spider-Man can do a low, side kick.
- Jumping Kick - While jumping in the air, Spider-Man can doing a side kick.
- Web Swinging - While jumping at a double height, Spider-Man will shoot out a diagonal web and swing along. This can only be done for a short time until the web-meter runs out.
- Web Shoot - While standing still, Spider-Man can shoot a glob of webbing from his wrists. This depletes his web-meter slightly.
- Wall Crawling - In the two vertical levels, Spider-Man will automatically be able to climb up the side of a building.
- Spider Sense - In the two vertical levels, Spider-Man will "buzz" with his spider sense, indicating that the player should move Spider-Man out of the way of possible falling objects.
- Web Vials - These items are used to restore Spider-Man's web-meter, since his webbing is in limited supply. They are dropped by henchmen throughout the levels.
- Hamburger - This tasty treat will restore some of Spider-Man's health meter.
- Point Bundles - When defeating a henchman, these bundles may drop and raise Spider-Man's point total.
- Health Meter - This is displayed at the bottom of the screen and shows how much life Spider-Man has left.
- Web Meter - Also displayed at the bottom of the screen, this meter shows how much webbing Spider-Man has left at his disposal.
- Alleyway - Receiving a call from Mysterio at his apartment, Spider-Man agrees to meet him in the alleyway. The level is linear, moving from left to right, and includes human henchman, as well as the Lizard popping up from manhole covers.
- To the Rooftop & Rooftop - Hobgoblin dares Spider-Man to meet him on the top of a skyscraper. The first part of the level plays out vertically, moving from bottom to top. Hobgoblin rains down explosives on Spider-Man, which can be averted by paying attention to his spider sense while dodging henchman attacking with baseball bats from windows. Once Spider-Man reaches the top, he can hop through a window and appear on the roof. On the roof, it becomes a normal left to right side scrolling level, with the Hobgoblin waiting at the end.
- Subway - Learning nothing from the Hobgoblin, Scorpion calls Spider-Man and tells him he will be waiting at the end of the subway line. As the level starts, Spider-Man is on the top of a subway car. Periodically, while the car is at rest, henchman will pull themselves up and shoot at him. Once Spider-Man makes his way past the station, the train starts up. On the way to the next station, bats will try to harm Spider-Man. After two stops, Spider-Man faces off against the Scorpion.
- Central Park - After no news from Scorpion, Rhino's call takes Spider-Man to Central Park. Besides the regular henchman and the lizard throughout the level, birds frequently fly about and briars fall from trees when Spider-Man steps in their vicinity. Near the end of the level, there is a transition back to an alleyway and Rhino is found at the end of one just like Mysterio from the first level.
- Skyscrapper & Rooftop #2 - The second vertical portion of the game, the first part takes you up another skyscraper, now with flying birds and henchmen who shoot at Spider-Man. On the second rooftop, birds fly about while Spider-Man makes his way to Doctor Octopus at the end.
- Sewer - In the final level, Spider-Man must fight his way through a sewer to battle Venom, the true kidnapper of Mary Jane. Crocodiles roam the fetid waters while henchmen fire on Spider-Man while hanging from water pipes. Also, waste from sewer drains impede his movement. At the end of the level, Spider-Man faces off against Venom. Upon Venom's defeat, Mary Jane and Spider-Man are reunited.