- Harry Osborn
- Stark Industries
- Norman Osborn (Main story and flashback)
- Doctor Octopus/Iron Octopus (Dr. Otto Octavius) (Main story and flashback) (First appearance as Iron Octopus)
- Deadpool (Wade Wilson) (On-screen)
Races and Species:
- Iron Man Armor
- Iron Octopus Armor (First appearance)
- Arc Reactor
- Iron Spider Armor
- Spider-Man's Web-Shooters
- Doctor Octopus' Tentacles
- Electroshock Gauntlet
Peter and Harry have games night at the Osborn penthouse, as his father had to work late and could not take them to a baseball game. Peter's spider-sense goes off and there is an explosion coming from Norman's office; when they rushed to check Peter gets trapped downstairs, telling Harry he will go for help, giving him time to change into Spider-Man. When Harry entered his father's office he finds him being attacked by Iron Man. Spider-Man arrives and tries to stop Iron Man who is non-responsive. He then tried to shoot him with missiles, but dodges them all. In the distraction Norman grabs a experimental machine gun and fires at Iron Man. During then he uses a electro-glove to disrupt the armor allowing Spider-Man to punch him knocking his head off much to Spider-Man horror. The armor is revealed to be empty, so he travels to Stark Industries to find out what happened. When he arrives he finds Iron Man battling his own armors; working with Spider-Man he takes them all out. They discover an Octobot hacking his system, which they try to follow but escapes through a vent. The Octobot returns to Doc Ock who uses the Arc Reactor technology to build his own armor. The Osborn are taken to the Helicarrier for protection, where they are called into Doc Connors lab to view his regarding a piece of the tentacle. Doc Connors explained that it was created by Dr. Otto Octavius. Osborn reveals that Octavius was a former employee that died in accident. Doc Ock reactivate's the tentacle and attack Osborn, but it is destroyed by Iron Man. Doc Ock in his Iron Octopus armor arrives and kidnaps both the Osborns. He takes them both to his secret lab and threatens Osborn. Spider-Man arrives in his Iron Spider Armor and rescues his friends. He then battled the Iron Octopus, but before he could defeat Spider-Man, Iron Man arrives and blast Doc Ock and is defeated due to his faulty Arc Reactor. Norman recovers Doc Ock's body and provides him the medical treatment he needs with plans to use him again in the future. Iron Man thanks Spider-Man and agrees to another possible team up in the future.
- Deadpool makes a cameo appearance in a video game.