Norman Osborn/Green Goblin (main antagonist)

Carnage's Faction [Carnage, Shriek, Carrion, Demogoblin and Spider-Doppelganger] (secondary antagonists)

Sinister Six (Doctor Octopus, Vulture, Electro, Mysterio, Sandman and Kraven the Hunter)

Doctor Eggman

Roman Torchwick

Nack the Weasel/Fang the Sniper (Bounty Hunter)

Femme Fatales (Cinder Fall, Emerald Sustrai, Neopolitan, Himiko Toga, Senki Shu and Silver Sable) [For now.]

Miguel O'Hara/Spider-Man 2099

Frieza (Final Boss)