While Mario is depicted as a hero throughout most of his history, he has been enemies with Donkey Kong and Donkey Kong Jr.. These are his villainous actions.
Coincidentally, Mario in his first game (the original Donkey Kong) was depicted as a naughty carpenter who abused his own pet, Donkey Kong. In the prequel Game & Watch game, Donkey Kong Circus, Mario forces poor Donkey Kong to perform at the circus by juggling pineapples and dodging fireball, and even laughs at him whenever he either gets hit by a fireball or drops a pineapple.
His role just worsened in the sequel, Donkey Kong Jr. as he is actually the villain in that game.
Donkey Kong Jr. (game)
Mario went on to imprison Donkey Kong in a cage. DK Junior went on to try and rescue his father. Mario attempted to stop the young ape and released various types of traps mainly consisting of animals. Junior eventually did make it to his father and the two returned to Donkey Kong Island just after Donkey Kong made a shot at Stanley the Bugman's greenhouse.
He also whipped the animals that worked for him, eventually known as Mario's slave labor. This makes him extremely evil and cruel, as he also let unnecessarily made them know who was in charge (him) his role was not better before as he abused DK before the events of Donkey Kong. Jr.
Donkey Kong Circus
Mario appears as the antagonist in Donkey Kong Circus. Mario is an audience member who laughs at Donkey Kong, the new attraction of the circus, whenever he drops a pineapple or gets hit by fire. In the end of the game, Mario is shown in the construction site where the first game begins.