Unlock Vampire Wario:
To unlock Vampire Wario as a playable character in Versus mode, complete Story mode on Normal or higher difficulty using Dr. Mario, but without losing a single time.

Unlockable Multi-player characters:
Metal Mario - Complete Story mode on Normal or higher difficulty using Wario, but without losing a single time.

Increase Computer Difficulty:
When selecting a computer opponent in Vs. Computer mode, hold the L button and press A to make his difficulty one star harder (the equivalent of Hard level).

By holding L and pressing B, the computer player's difficulty increases two stars (the equivalent of S-Hard level).

S-Hard Difficulty:
To open up S-Hard difficulty in Story mode, highlight Hard and press the Z button. Pressing the Z button again switches it back to Hard.

More Viruses:
To get more Viruses, Press L or R in marathon mode to increase the number of viruses.