First appearance of Bowser Jr, Piantas, Nokis, and Cataquacks.
The FLUDD was created by E. Gadd Industries. This is the same company that created the Poltergust vacuum in Luigi's Mansion (2001).
Isle Delfino is shaped like a Dolphin. "Dolphin" was the original unofficial name given to the Nintendo Gamecube.
In the attic of the Hotel Delfino, the janitor complains about the ghosts, wishing someone would come and "suck them up in a vacuum cleaner". This is a reference to another Gamecube title, Luigi's Mansion (2001), where Luigi did indeed suck up ghosts in a vacuum cleaner.
This was Mario's first official game for the Nintendo GameCube.