Villains: Norman Osborn/Dark Goblin • John Jameson/Man-Wolf • Ollie Osnick/Steel Spider • Spencer Smythe • Symbiote • Brock Rumlow/Crossbones • Abner Jenkins/Beetle • Otto Octavius/Doctor Octopus/Superior Spider-Man • Raymond Warren/The Jackal • Ghost • Herman Schultz/Shocker • Aleksei Sytsevich/Rhino • Felicia Hardy/Black Cat • Keemia Marko/Sandgirl • Hammerhead • Sergei Kravinoff/Kraven the Hunter • Carl Creel/Absorbing Man • Arnim Zola • Phineas Mason/Tinkerer • Jack O'Lantern • Francine Frye/Electro • Mark Raxton/Molten Man • Carolyn Trainer/Lady Octopus
Other characters: Flash Thompson/Venom • Aunt May • Ben Parker • Max Modell • Anna Maria Marconi • Harry Osborn/Hobgoblin • Mary Jane Watson • Maria Corazon

Season Two: "How I Thwipped My Summer Vacation" • "Take Two" • "Between an Ock and a Hard Place" • "Rise Above It All" • "School of Hard Knocks" • "Dead Man's Party" • "Venom Returns" • "Bring on the Bad Guys" • "Brain Drain" • "The Living Brain" • "The Day Without Spider-Man" • "My Own Worst Enemy" • "Critical Update" • "A Troubled Mind" • "Cloak and Dagger" • "Superior" • "Brand New Day" • "The Cellar" • "The Road to Goblin War" • "Goblin War"
Season Three: "Web of Venom" • "Amazing Friends" • "Vengeance of Venom" • "Spider-Man Unmasked" • "Generations" • "Maximum Venom"