Chicken
Successfully finish Story mode with all characters and accumulate 10 hours of game play time to unlock Chicken.

Eris
Successfully finish Story mode with Billy, Mandy, Grim, or Irwin to unlock Eris.

Mogar
Successfully finish Story mode with Billy, Mandy, Grim, or Irwin to unlock Mogar.

Jack O'Lantern
Successfully finish Mission 9 in Tier 2 in Mission mode to unlock Jack O'Lantern.

Concept art
From MAIN MENU, hold Circle and press Up(2), Down(2), Left, Right, Left, Right to unlock concept art.

Nergal
Successfully finish Story mode with Billy, Mandy, Grim, or Irwin to unlock Nergal.

General Skarr
Successfully finish Mission 6 in Tier 4 in Mission mode to unlock General Skarr.

Dracula
Complete Mission 5 in Tier 3 in Mission mode to unlock Dracula.

Hoss Delgado
Complete Mission 9 in Tier 1 in Mission mode to unlock Hoss Delgado.

Nerglings
Successfully finish Story mode with all characters to unlock Nerglings.

Fred Fredburger
Successfully finish Story mode with Billy, Mandy, Grim, or Irwin to unlock Fred Fredburger.

Lord Pain
Successfully finish Mission 9 in Tier 5 in Mission mode to unlock Lord Pain.

Vikings
Successfully finish Story mode with all characters and destroy every letter in the credits sequence to unlock Vikings.

Boogey
Successfully finish Mission 9 in Tier 4 in Mission mode to unlock Boogey.

Alternate costumes/outfits
Successfully finish Story mode with any character to unlock an alternate costume/outfit for that person.

Nergal Jr.
Successfully finish Mission 9 in Tier 3 in Mission mode to unlock Nergal Jr.