A list of characters from Spider-Man: The Animated Series.
- Main Characters note Peter Parker / Spider-Man, May Parker, Mary Jane Watson, J. Jonah Jameson, Felicia Hardy / Black Cat
- Supporting Cast note Dr. Curt Connors / The Lizard, Harry Osborn / Green Goblin II, Joseph "Robbie" Robertson, Anna Watson, Flash Thompson, Terri Lee, Debra Whitman, Liz Allan, Anastasia Hardy, Farley Stillwell, Taina, Ashley Kafka, Gwen Stacy
- Villains note Wilson Fisk / The Kingpin, Spencer Smythe, Alistair Smythe, Herbert Landon, Dr. Otto Octavius / Dr. Octopus, Mac Gargan / The Scorpion, The Rhino, The Shocker, Quentin Beck / Mysterio, Adrian Toomes / The Vulture, The Chameleon, Eddie Brock / Venom, Cletus Kasady / Carnage, The Hobgoblin, Norman Osborn / The Green Goblin, Silvio "Silvermane" Mafredi, Alisa Silver / Alisa Silvermane, Hammerhead, Lonnie Lincoln / Tombstone, Morrie Bench / Hydro-Man, Baron Mordo, Dormammu, Miles Warren, Rheinholt Kragov / Electro, The Red Skull, Victor Von Doom / Doctor Doom, Miriam, Dr. Jonathan Ohnn / The Spot, Hobie Brown / Prowler, Richard Fisk, Susan Choi Jackson Weele / Big Wheel
- Allies and Other Heroes note Madame Web, Michael Morbius, Sergei Kravinoff / Kraven the Hunter, Dr. Mariah Crawford, Silver Sable, The Fantastic Four, Stephen Strange / Doctor Strange, Blade, Abraham Whistler, The Punisher / Frank Castle, Nick Fury, Tony Stark / Iron Man, James Rhodes / War Machine, Matt Murdock / Daredevil, Steve Rogers / Captain America, The X-Men, The Beyonder, The American Six, Robert Farrell / Rocket Racer
- Alternate Spider-Men note Iron Spider-Man, Six Armed Spider-Man, Mechanical Armed Spider-Man, Unknown Spider-Man, Ben Reilly / The Scarlet Spider, Spider-Carnage