If you play at Peach's Castle and your golf ball lands in a yellow flower bed, the 3 original types of Pikmin will fly out.

Luigi signs an L-shape multiple times in the game which fans believed to be the American Sign Language word for lesbian. Luigi is not actually signing the word 'lesbian', as he is using the wrong hand, and is pointing in the wrong direction.

If the player doesn't hit the ball for a while, Boo will appear with helpful information and tips. If the player doesn't hit the ball for a while longer however, Boo will then taunt them with various messages, including:
•Why haven't you hit the ball yet?
•Do you plan on hitting the ball....TODAY?
•You're a slow player!
•Hey, are you even there anymore?
•Hey, you've got to hit the ball to play!
•What!? You want to go the the clubhouse?
•I don't want to wait forever!
•Hit it already!

In the North American release of the game, during one of the Mario Golf: Toadstool Tour tutorials, one of Toad's tips is misspelled, with "If you press B" coming out as an unintentional profane message: "f you press B". This typo is not present in other regional translations of the game and is corrected in the European English releases.