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Prior to the events of the game, Mario, Luigi, Yellow Toad, Blue Toad and Yoshi were visiting Princess Peach in her Castle. As soon as they proceed to leave, Mouser appears, along with his troops, and abducts all said characters.
After getting through eight worlds, Mario and co. will arrive at Mouser's Hideout. Mouser will throw Bob-ombs and the player should throw them back at him. When this goes successfully five times, Mouser will be defeated. However, his most loyal servants, other bosses the player had fought, arrive and use a special sceptre to turn Mouser giant. A level will play which involves Mouser chasing the player. At the end waits an enormous button. When pressed, all the poison beneath will disappear and Mouser and his servants will fall down.
Koopa Troopas are basic enemies. When the player stands upon it, it will retreat into its shell, which can be picked up and thrown away, which will result in it sliding. When it hits another enemy, it will defeat it and when it hits a wall, it will bounce back. Note that the Koopa Troopas can't be picked up when they are out of their shells.
Phantos are mask-like creatures who will chase Mario and co. when they enter the room the Phantos are in. Phantos can't be picked up and thereby don't have any weight.
Pokeys are tall enemies whom will damage the player upon contact. Their amount of body segements differs per Pokey. They also sometimes slide back and forth slowly.
Pansers are fire-spitting enemies. When the player gets close to them, they will release three fireballs. They can walk back and forth, however, some don't move at all. It is the only enemy who is invincible against Iceballs. They also can't be picked up.
These enemies fly through the air, sometimes trying to hinder players. They attack from the air sometimes as well, by throwing bombs. The player can jump on it to make it descent downwards.
An Ostro is an enemy that runs towards the player. The player can jump on it and steer it past a lot of obstacles, seeing as they can jump with it as well. It can be picked up, and has a small amount of weight.
Trouters are fish that jump up and down on waterfalls. They can be jumped on by the player, but if they stand upon it even when it falls back to the depths of where it came from, they will lose a life. They can't be picked up.
Cheep-Cheeps are enemies that swim underwater. In some levels with a water surface, they sometimes jump out, similar to Trouters. The player can't stand on them.
Whales are large creatures that appear in watery areas sometimes. They can be ridden by the player over the water. They will be hurt if they touch it from the side of when they are hit by its tail. They cannot be picked up by the player. Their spout can be used effectively to reach higher areas.
Tryclyde will attack by jumping on the player and shooting black balls towards them. The player needs to throw itmes at him to make him confused, and then stomp on one of the three heads to do damage. This has to be done three times, due to the boss having three heads.
Fryguy attacks by shooting Fireballs at the player. When the player happens to touch him, they would receive damage. Like all other bosses, he can't be picked up. The player needs to throw items at him five times.
Mouser is the main enemy of the game. In his first battle, he throws Bombs at the player, which they should pick up and throw back at him before they explode. In the second battle, He grows giant with the help of the other bosses and a magic wand, and he'll follow Mario and co. through a level full of poison which he walks through. If the player touches the poison, they will lose a life instantly. At the end of the level, a large button waits to be pressed and when it is pressed, all the poison and ground beneath Mouser disappears and the player completes Story Mode.
The Ice Flower turns the character into their Ice form. They can throw Iceballs which will encase most enemies into a block of Ice, which can be sliden away much like a Koopa Troopa.
The Super Leaf turns the characters into their Raccoon form. In this state, they can use their tail as way to attack and if they run for five seconds, a bar will fill up and they can fly for a short amount of time. They can also glide.
The Stopwatch Flower will turn the characters into their Stopwatch form. In this state, they can throw a Stopwatch at an enemy, which will making it stand still at the same place for a few seconds.
The Starman will turn the player into their Invincible form. In this state, they are harmless to enemies and will defeat most of them upon contact. Its effect will only last for ten seconds.
The Mini Mushroom will transform the characters into their Mini from. They will be extremely small, which enables them to walk on water, jump extra high and travel down Mini Pipes. However, the Mini Mushroom also has its downsides; the player will lose a life when hit and is unable to pick up Mediumweight or Heavyweight items or enemies.
The Mega Mushroom will turn the player into their Mega form. In this state, they can crush almost anything except for solid ground and Pipes which are able to enter. Said Pipes will be pushed to shorter size, however.
The Super Acorn will transform the characters into their Flying Squirrel form. In this state, they can glide slightly, cling to walls and preform a mid-air jump.