Dr. Mario 64

Super hard difficulty:
Select story mode, highlight the "Hard" difficulty setting, then press Z.
Information in this section was contributed by energ_x1.

Expert mode:
Select versus mode, highlight a CPU player, then hold L and press A (for Hard difficulty) or B (for Super hard difficulty).
Information in this section was contributed by energ_x1.

More viruses:
Press L or R in marathon mode to increase the number of viruses.

Metal Mario:
Successfully complete story mode as Wario under the normal, hard, or super hard difficulty setting without losing or using the redo option. Defeat Metal Mario without losing to unlock him in versus mode.

Vampire Wario:
Successfully complete story mode as Dr. Mario under the normal, hard, or super hard difficulty setting without losing or using the redo option. Defeat Vampire Wario without losing to unlock him Mario in versus mode.

Hint: Super Mario 64 reference:
When playing Story mode as Wario, if you play against Metal Mario the number of viruses will be 64. The game refers to the character as "Metal Mario", whose first appearance was in Super Mario 64.
Information in this section was contributed by Augusto.

Hint: Dr. Mario reference:
When playing any VS mode (Story, Vs Computer, Two Player Vs or Multi-player), you will hear two combo songs from the first Dr. Mario. In Classic mode you can only hear one of them. Sometimes, you can also hear the 1-Up music from the Mario game series.
Information in this section was contributed by Augusto.