Peter Parker/Spiderman
Josh Keaton
Gwen Stacy
Lacey Chabert
Flash Thompson (voice) / Flash Thompson / Car Thief #1 (voice)
Joshua LeBar
Betty Brant/Sally Avril
Grey Griffin
Liz Allan (voice) / Liz Allan / Trina (voice)
Alanna Ubach
Mary Jane Watson (voice) / Mary Jane Watson / Rosie Thompson (voice)
Vanessa Marshall
Harry Osborn (voice) / Harry Osborn / Frederick Foswell (voice)
James Arnold Taylor
Bernard Houseman
Jeff Bennett
Curt Connors (voice) / Curt Connors / Lizard (voice)
Dee Bradley Baker
Hammerhead (voice) / Sandman / Flint Marko
John DiMaggio
Tinkerer
Thom Adcox-Hernandez
Vulture (voice) / Adrian Toomes
Robert Englund
Eddie Brock (voice) / Venom (voice) / Eddie Brock
Ben Diskin
Miles Warren
Brian George
May Parker (voice)
Deborah Strang
Jean DeWolff
Irene Bedard
Electro (voice) / Max Dillon / Thug #1 (voice)
Crispin Freeman
Green Goblin/Chameleon
Steve Blum
George Stacy (voice) / Rhino / Alex O'Hirn (voice)
Clancy Brown
Otto Octavius (voice) / Doctor Octopus (voice) / Master Planner (voice)
Peter MacNicol
Norman Osborn (voice)
Alan Rachins
Quentin Beck/Mysterio
Xander Berkeley
Silver Sable
Nikki Cox
Rand Robertson (voice) / Rand Robertson / Robbie Robertson (voice)
Phil LaMarr
J. Jonah Jameson (voice) / J. Jonah Jameson / John Jameson (voice)
Daran Norris
Tombstone (voice) / Principal Davis (voice) / Tombstone
Kevin Michael Richardson
Glory Grant (voice) / Glory Grant / Cobweb
Cree Summer
Black Cat (voice) / Felicia Hardy
Tricia Helfer