- Josh Keaton
Peter Parker / Spider Man
- Deborah Strang
May Parker
- Vanessa Marshall
Mary Jane Watson
- Lacey Chabert
Gwen Stacy
- Clancy Brown
George Stacy
- James Arnold Taylor
Harry Osborn
- Daran Norris
J. Jonah Jameson
- Alanna Ubach
Liz Allan
- Grey Delisle
Sally Avril, Betty Brant
- Irene Bedard
Jean DeWolff
- Joshua LeBar
Eugene „Flash” Thompson
- Kath Soucie
Anna Watson, Martha Connors
- Phil LaMarr
Robbie Robertson, Randy Robertson
- Andrew Kishino
Kenny „King” Kong
- Dee Bradley Baker
Dr. Curt Connors/Lizard
- Robert Englund
Adrian Toomes/Vulture
- Peter MacNicol
Otto Octavius/Doctor Octopus
- Alan Rachins
Norman Osborn
- Steve Blum
Green Goblin
- Crispin Freeman
Max Dillon/Electro
- Ben Diskin
Hammerhead
- Clancy Brown
Rhino
- Jeff Bennett
Montana, Shocker
- John Dimaggio
Flint Marko/Sandman, Eddie Brock/Venom
