Josh Keaton

Peter Parker / Spider Man (voice)

Lacey Chabert

Gwen Stacy (voice)

Joshua LeBar

Flash Thompson (voice), Flash Thompson / Car Thief #1 (voice)

Grey DeLisle

Voice of OsCorp (voice), Sally Avril (voice), Sally Avril / Bette Brant (voice), Sally Avril / Voice of OsCorp (voice), Sally Avril / Erin (voice), Sally Avril / Betty Brant (voice), Sally Avril / Coffee Clerk (voice), Sally Avril / Computer Voice (voice), Stephanie Briggs (voice), Betty Brant (voice)

Alanna Ubach

Liz Allan / Trina (voice), Liz Allan (voice), Liz Allan / Helena (voice)

Vanessa Marshall

Mary Jane Watson (voice), Mary Jane Watson / Rosie Thompson (voice), Mary Jane Watson / Hermia (voice)

James Arnold Taylor

Harry Osborn (voice), Harry Osborn / Patch / Frederick Foswell (voice), Harry Osborn / Alan O'Neil (voice), Patch / Homunculi (voice), Harry Osborn / Frederick Foswell (voice)

Deborah Strang

May Parker (voice)

Clancy Brown

Alex O'Hirn / Ox (voice), George Stacy (voice), Rhino / George Stacy (voice), Alex O'Hirn / Rhino / George Stacy (voice), Alex O'Hirn (voice)

Alan Rachins

Norman Osborn (voice)

Phil LaMarr

Rand Robertson / Robbie Robertson (voice), Robbie Robertson (voice), Fancy Dan / Homunculus #1 (voice), Fancy Dan / Ricochet / Rand Robertson (voice), Rand Robertson / Fancy Dan (voice), Rand Robertson (voice)

Ben Diskin

Eddie Brock / Venom (voice), Eddie Brock (voice), Venom (voice)

Daran Norris

J. Jonah Jameson / John Jameson (voice), J. Jonah Jameson (voice), J. Jonah Jameson / John Jameson / Colonel Jupiter (voice)

Kevin Michael Richardson

Coach Smith (voice), Big Man / Coach Smith / Thug (voice), Tombstone (voice), Tombstone / Principal Davis (voice), Principal Davis (voice), OsCorp Security Guard (voice), Big Man / Tombstone / L. Thompson Lincoln (voice)

Peter MacNicol

Master Planner / Otto Octavius (voice), Doctor Octopus (voice), Master Planner (voice), Otto Octavius / Doctor Octopus (voice), Otto Octavius (voice), Master Planner / Doctor Octopus (voice)

John DiMaggio

Sandman (voice), Flint Marko / Hammerhead (voice), Sandman / Hammerhead (voice), Flint Marko / Sandman / Hammerhead (voice), Hammerhead / Car Thief #2 (voice), Hammerhead (voice)

Dee Bradley Baker

Curt Connors / Homunculi (voice), Curt Connors (voice), Curt Connors / Lizard (voice)

Cree Summer

Glory Grant / Cobweb / Homunculus #2 (voice), Glory Grant (voice)

Crispin Freeman

Electro / Clerk (voice), Electro (voice), Thug #1 (voice), Max Dillon / Electro (voice)

Robert Englund

Vulture (voice), Adrian Toomes / Vulture (voice)

Dorian Harewood

Doc Bromwell (voice)

Tricia Helfer

Felicia Hardy / Black Cat (voice), Black Cat (voice)

Miguel Ferrer

Silvio Manfredi / Silvermane (voice), Silvermane (voice)

Eric Vesbit

Sergei Kravinoff / Kraven the Hunter (voice), Kraven (voice)

Ed Asner

Ben Parker (voice)

Danny Trejo

Ox (voice)

Stan Lee

Stan (voice)

Steve Blum

Dilbert Trilby / Seymour O'Reily / Green Gobilin (voice)