Josh Keaton (29)

Peter Parker / Spider Man (voice)

Ed Asner (78)

Ben Parker (voice)

Grey DeLisle (34)

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

Phil LaMarr (41)

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

Cree Summer (38)

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

Kevin Michael Richardson (43)

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

Tricia Helfer (33)

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

Robert Englund (60)

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

Crispin Freeman (36)

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

Peter MacNicol (53)

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

Deborah Strang (57)

May Parker (voice)

Dee Bradley Baker (45)

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

John DiMaggio (39)

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

Ben Diskin (25)

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

Clancy Brown (49)

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

Joshua LeBar

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

Vanessa Marshall (38)

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

Daran Norris (43)

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

James Arnold Taylor (38)

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

Alan Rachins (65)

Norman Osborn (voice)

Alanna Ubach (32)

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

Lacey Chabert (25)

Gwen Stacy (voice)

Miguel Ferrer (53)

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

Danny Trejo (63)

Ox (voice)

Stan Lee (85)

Stan (voice)

Eric Vesbit

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

Dorian Harewood (57)

Doc Bromwell (voice)

Steve Blum (47)

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