Otto Octavius, a brilliant physicist known to his colleagues as Doctor Octopus, is conducting experiments using four mechanical arms connected to his body. An incident in an atomic plant fuses his arms to his body and causes the man to go mad with power. While he is holding hospital employees captive, Spider-Man tries to tackle him but Dr. Octopus easily defeats him and tosses him out of the window. Shortly after taking complete control of the plant, Dr. Octopus sees that Spider-Man is back on the surveillance monitors but the hero soon disappears from his scanners. The hero defeats him by melting his mechanical arms together with a chemical and by blinding him with web-fluid. The hero leaves him tied up for the authorities to pick up.

Doctor Octopus has served his sentence and is being released from prison earlier for good behavior. Betty Brant comes to pick him up and they drive to Philadelphia, as it was planned. There, Doctor Octopus breaks Blackie Gaxton out of jail. But instead of releasing Bennett Brant from his gambling debt, as he said he would, Gaxton takes him and his sister Betty hostage aboard his getaway boat. Octopus joins him to get payment for his service. Spider-Man finds them all and a battle ensues. After a long fight, Doctor Octopus is able to escape, but the police arrive to pick up Blackie and his gang. Then he goes on a crime spree in Philadelphia to get Spider-Man to fight him again, but ultimately he has to capture Betty Brant in New York to catch the hero's attention. Surprised that Spider-Man is much weaker than usual, he knocks him out and unmasks him to find out that it is only Peter Parker. Next day, he breaks loose many beasts from the city zoo to attract the hero again, but this time the web-crawler is more than a match. Octopus gets trapped in a blazing building and taken away by the police.

Doctor Octopus breaks out of prison and forms the Sinister Six with Electro, the Vulture, Sandman, Kraven the Hunter and Mysterio to get rid of Spider-Man once and for all. Following his plan, Electro and Sandman kidnap Betty Brant and May Parker and surprisingly, Doctor Octopus, who is holding them hostage, treats the ladies like a gentleman. When Spider-Man finds his castle, Octopus traps him in a giant fishbowl inside which he fights him, but the hero entangles him in webbing before calling the police and getting away with May and Betty.

Behind bars, Octopus is jealous of the Green Goblin's victory over Spider-Man.