- Sinister Six (First appearance)
- Daily Bugle Communications
- J. Jonah Jameson (Appears on a computer screen, TV, or hologram only)
- Flash Thompson (Vision)
The team trains on Liberty Island, when Spider-Man is called by Doc Connors to investigate Doc Ock's hidden base. Where Doc Ock has Doc Connors trapped as well. He confronts Doc Ock but he had brought some allies, five of his most dangerous foes, including Rhino, Kraven the Hunter, Electro, Beetle, who introduced as his "Sinister Six". Spider-Man confused as their was only five, but Doc Ock reveals the Lizard is the Sixth member and mutates Connors into he alter ego. He battled the six criminals but barely escaped into the sewer. The six tracked him, but split up allowing Spider-Man to capture the Lizard and revert him back. But Ock arrived and reverse the change and the Lizard escaped. With Spider-Man now captured they argued on who gets to kill him. Spider-Man escapes and leads them to the shipping yard, where they are filmed by the Daily Bugle Helicopter. Using the camera he called for help from the rest of the team. Fighting alone dodging each attack long enough, for everyone else to arrive. The two teams then battled, as they fight Spider-Man battles the Lizard who is now free from Do Ock's control escapes into the sewer. Spider-Man returns to the surface to destroys Doc Ock's robotic arms. The five criminals are then taken in custody by S.H.I.E.L.D.
- The Sinister Six appear in the "sixth" episode of the second season.
- The villains that make up the six lead by Doctor Octopus are the villains featured in season two episodes leading up to the sixth episode. Lizard, Electro, Rhino, Kraven, and then Beetle was the main villain in the episode titled Hawkeye.
- Sinister Six first appeared in the comics in issue Amazing Spider-Man Annual # 1. Doctor Octopus, Electro and Kraven the Hunter are part of the original team.