When playing co-op, a player that loses all their lives during a level while the other player(s) is/are still alive can hold different directions on the D-Pad and then press 1 or 2 to play specific sound effects over the level's music like a snare drum, kick drum, cowbells, clapping, and two jump-like sound effects.

A local game retailer in Australia released the game on November 6, 2009, six days prior to the official Australian release date. 24-year old James Burt bought the early copy of the game and shared it with others on a file-sharing network, where people could illegally download the game. Despite the 50,000 downloads, Nintendo of Australia later took it down and sued James in court for copyright infringement on February 6, 2010. He was forced to pay AU$1.5 million.

The game's files contain an unused fanfare sound, named "cheepfanfare_lr.ry.32"

The file "obj_block.arc" not only contains an early design for the block roulette, similar to the one in New Super Mario Bros., but it also includes early icons for the powerups as seen on the left. Note the change of Mini Mushroom to a 1UP in the final, and lack of Ice Flower in the early design.

Around every 30 Seconds of World 9's Map theme, a segment from Mario Kart 64's Rainbow Road theme will play.

When enemies dance in sync with both games' music, Pokeys will swap their spikes for flowers for a brief moment. If a Pokey is eaten with Yoshi during this point in time it will be instantly defeated and the Yoshi will lay an egg containing a power-up. The power-up can range from coins to 1-ups depending on how many segments the Pokey had when it was eaten.

Shigeru Miyamoto considered making Princess Peach a playable character, like in Super Mario Bros. 2, instead of the damsel in distress. He eventually decided against it because of how difficult it would have been to animate her dress, and instead had two Toads. They were eventually able to manage having Peach being playable in mainline games with Super Mario 3D World, Super Mario Run and Super Mario Bros. Wonder.

The game was originally planned to have a chicken suit power-up. It is unknown what it would have done, and is thought to have been replaced by the Penguin Suit.

By getting 99 lives at any point in the game with Mario, he will start playing with his hat missing until he loses a life. This is just cosmetic as it changes nothing to the gameplay. This also only works with Mario as collecting 99 lives with any other character does nothing to them.

On the disc, the characters' colours are arranged in the same pattern as the buttons on the Japanese and PAL SNES controller. It's unknown if this was intentional.

There is a cactus prop at Universal Studios' Super Nintendo World that does not originate from an official Mario game, but rather the fanmade mod Newer Super Mario Bros. Wii. At the time of the park's opening, a Google search for "new super mario bros wii cactus" would show images of Newer's desert levels alongside New Super Mario Bros. Wii, meaning that the cacti was likely added by accident, being mistaken for official Nintendo material.