If the player performs a set of Bros. Attacks correctly Mario will get an idea to do an Advanced Bros. Attack, illustrated with a small thinking animation. If this occurs during the battle against the Hoohooros boss while said boss is also using the thinking animation, the game will soft lock.

In the later-released Japanese version, Heart Blocks were added to Beanbean Castle Sewers, Oho Oasis, and Teehee Valley; these blocks fully restore Mario and Luigi's HP and BP at no cost. These blocks also feature in the 3DS remake across all regions, as the remake is based on the Japanese version of the original game.

If Bowser defeats Mario at the very start of the game, it will soft lock as the developers never intended for Mario to lose.

The game has an unused background that uses graphics from Wrecking Crew.

The games contains graphics for an unused cloaked character.

In the Japanese version, Boomerang Bros are capable of dropping an item called "Oho Gear". This item raises defense by 28 points and can be equipped to either Mario or Luigi. For unknown reasons, it was removed from all International ports of the game. However, the item can be found in the game's coding and is only obtainable through cheating devices. The item is fully functional in all versions and its description, "Pants with a raging wave print," was translated into multiple languages in the game's original release. This seems to indicate that the Oho Gear was originally meant to be in the game's international releases, but was disabled late in its development.

During the first tutorial of Border Jump, if Mario jumps when the Border Bros. command Luigi to jump, they will tell Mario to "give the 'Jumpman' thing a rest". This is a nod to "Jumpman", Mario's original name used in the Japanese version of Donkey Kong.

There are three unused songs in this game. The first is never used for anything, the second and third, however are variants of the main theme from a Japan-only game known as Tomato Adventure, another one of AlphaDream's games.

When the brothers receive the Hammers from the Sledgehammer Brothers, one of them says "Pow! Now you're playing with power!" which is one of Nintendo's slogans.

If Popple is defeated before Birdo in his final fight, Birdo says: "You may have defeated my darling, but this is as far as you go!" as a reference to her quote in Super Mario Advance: "This is as far as you go

Inside the game's code are unused button and ability icons. The final "bubble" sprites are much larger, and the final icons are bigger and more detailed.

A number of Nintendo characters from other games were originally going to appear in the Starbeans Café. Captain Olimar, Samus Aran, Fox, Wario, an Excitebike Racer, and Link would have shown up and each given the player an item. E. Gadd would only have shown up on the player's first visit to the coffee house. The items the player receives were also different.

• Olimar: UV Lamp, replaced with Cobalt Necktie
• Wario: Wario's Gold, replaced with Greed Wallet
• Samus: Energy Tank, replaced with Power Grip
• Excitebike racer: Excite Spring
• Link: Triforce, replaced with Great Force
• Fox: Gold Ring, replaced with Bonus Ring
• E. Gadd: Game Boy Horror, replaced with Game Boy Horror SP

The names of all of the Beanbean Kingdom locations, except for Yoshi Theater and Little Fungitown, contain words pertaining to laughter.

• Hoohoo Village
• Hoohoo Mountain
• Chucklehuck Woods
• Woohoo Hooniversity
• Teehee Valley
• Guffawha Ruins
• Oho Oasis
• Oho Ocean
• Oho Ocean Seabed
• Gwarhar Lagoon
• Joke's End

The battle backgrounds are actually 512 pixels wide by 256 pixels tall. However, only a 240x160 pixel area is visible during play, a little more than 25% of the total image. It's possible that they were intended to scroll during battle. The final battle has some scrolling, and the concept was later used in Mario & Luigi: Bowser's Inside Story.

In the Yoshi Theater, there are four posters advertising Nintendo characters. Besides Mario and Luigi, the posters show Kirby, Wario, and a (then) Japanese only character, Stafy.

This is the first game after Hotel Mario that features the Koopalings, the bosses of each world in Super Mario Bros. 3 and Super Mario World.

This is one of the few Game Boy Advance titles to use the rumble effect of a GameCube controller when being played on a Game Boy Player.

There is a reference to Donkey Kong in the Woohoo Hooniversity. When Luigi is taken by the cairn, Mario has to run up a series of ramps with barrels rolling down at him.

Geno from Super Mario RPG: Legend of the Seven Stars makes a cameo in the Star 'Stache Smash minigame.