In the English version of the game, Bowser's laugh in the opening has a noticeable echo to it. This is oddly absent from the French release of the game.
Contributed by billebobfacts
There is an unused help dialog that uses the same voice for Mario as the unused cutscene for when the player defeats Roy.
Contributed by xianc78
An early version of the cutscene that plays when you beat Roy doesn't use the voice actors from the final game. The voices of the Mario brothers seem to be heavily based on Walker Boone and Tony Rosato's takes on the brothers from the Mario cartoon series airing at around the same time. Oddly enough, this is stored in the data for the final level, not Roy's. There is no known cutscene data.
Contributed by billebobfacts
Iggy Koopa is the only koopa without a hotel. However, looking through the game's data reveals each hotel has an internal music, graphics and enemy data file labeled from L1 to L8. L6 is missing in its entirety, and the game jumps from Ludwig's to Wendy's hotel (L5 to L7).

You can extract sound and cutscene data from L6, but the hotel is left entirely blank. This points toward Iggy being planned to have a hotel of his own, opposed to only appearing in Bowser's hotel.
Contributed by Outofmind23
Depending on the day the CD-i's clock is set to, the "HERE WE GO" screen will be replaced with a different message. For example, if the clock is set to January 1st, the message would be "HAPPY NEW YEAR".
Contributed by pyoro64