"The New Adventures of Spider-Man" is a retelling of the popular "Spider-Man" comic book series, with twists and turns put in, as well as a surprise twist at the end.
- Peter Parker/Spider-Man: A young high school student and social outcast, he was bitten by a radioactive spider, and became "Spider-Man".
- Daredevil/Matt Murdock: A blind vigilante, he poses as a lawyer, and helps Spider-Man fight the Kingpin.
- The Thunderbolts: A group of reformed supervillains that aid Spider-Man and Daredevil in guarding New York.
- Songbird/Melissa Gold: The current leader, she can release sonic waves from her mouth.
- Paladin: Once a bounty hunter, him and Bullseye have "history". Uses weapons to fight, including grenades and guns.
- Ant-Man/Eric O'Grady: A perverted young man who can make himself shrink, he uses his powers for personal gain, and looks up to Spider-Man.
- Justice/Vance Astrovik: A telepath that accidentally killed his father, he aides the Thunderbolts in battle now.
- The Kingpin/Wilson Fisk: An obese crime lord, he has hired many thugs and low-life hoods to kill Spider-Man, but has failed many times.
- Green Goblin: A mysterious goblin-themed crook, he uses pumpkin bombs and destructive tools to destroy New York. Connected to OsCorp.
- The Scorpion/Mac Gargon: Jonah Jameson used him to try and kill Spider-Man, but he was merged with a bionic suit, and now wants nothing more than to kill Jameson.
- Dr. Miles Warren: A scientist that works for Norman Osborn, he is deranged and has tried making an army of Green Goblin clones, but they all failed. Currently in jail.
- Curt Connors/The Lizard: A scientist, he experimented with lizard DNA, and became a savage beast.
- Kraven the Hunter: A savage hunter, he has a primal sense and relentlessly hunts down Spider-Man in a game of "cat and mouse".
- Calypso: Kraven's lover, she uses special potions and tribal techniques to control animals.
- The Sandman/Flint Marko: A criminal who was trapped in an experiment that resulted in his body turning into sand.
- The Rhino/Aleksei Sytsevich: Another criminal, he was attached to a powerful suit, he is berserk and wants revenge on OsCorp.
- Speed Demon/Jimmy Sanders: A former pro-racer nicknamed "The Whizzer", he has a love for speed and gets an insane thrill out of speeding and mass destruction.
- Shocker/Herman Schultz: A criminal with a powerful bio-suit, he uses gauntlets to shoot electricity.
- Menace: A demonic bank robber, his true identity is unknown.
- Dennis Carradine: A middle-aged burglar, he killed Peter's Uncle Ben.
- Mary Jane Watson: Peter's neighbor and love interest for much of the series.
- Aunt May: Peter's elderly aunt, and his legal guardian after the deaths of his parents.
- Uncle Ben: Peter's uncle, he was shot dead by Dennis Carradine, resulting in Peter's transformation into Spider-Man.
Peter Parker learns a great lesson after his Uncle Ben dies, and he sets out to catch the killer, meanwhile remembering how he had become the "Spider-Man".
Peter becomes an intern at a lab, where a scientist turns into the savage "Lizard". Peter and the Lizard fight, and the Lizard is injected with a vaccine.
Spider-Man apprehends a crook, who later escapes from jail and is offered ultimate power by Norman Osborn. However, in the aftermath he is turned into the Sandman. Meanwhile, Peter tries to get a job at the Daily Bugle. Nearly destructible, Spider-Man only beats him by bursting a water tower on him, washing him into the sewers.