Unlock Metal Mario
Set the game on normal/hard/superhard difficulty and finish it without losing a single time using Wario. When you get up to, and defeat Rudy, you will then come face to face with Metal Mario!!! You have to defeat him without losing at all (you cannot even Pause and "Redo"), and then he will be unlocked in the Vs Modes.

Unlock Vampire Wario
As above, except you have to do all this with Dr. Mario, and at the end you will fight Vampire Wario. Remember you cannot lose a single time.

Unlock Super Hard mode
Go to story mode and highlight the Hard difficulty setting. Now press Z and you will change this setting to Super Hard.

Difficulty setting in Vs Mode
From the Character Selection Screen in Vs Mode, highlight any character and Hold L. You can then press A, or B to change the computer controlled character to 1 star (Hard) or 2 stars (Super Hard), respectively.