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The Amoeba Boys are a trio of sentient, man-sized bacteria who roam the streets of Townsville. Their biggest desire is to become well recognized criminals in the underworld. They often try to earn a notable reputation by attempting to fight the Powerpuff Girls, who simply shrug them off as not threatening and therefore not in need of their attention.

After years and years of trying and failing to get the Powerpuff Girls to pay attention to them, the Amoeba Boys would try and get the attention of the new hero Spider-Man, but would often not have much success with the web-slinger either ignoring or simply webbing them to a lamppost. The Amoeba Boys would finally earn their "hardcore" reputation, however, when they were enlisted by Jason Macendale, the Hobgoblin, to help capture Spider-Man under the orders of Princess Morbucks. When the four succeeded in capturing Spider-Man, however, the plot to hold the hero on "trial", with the jury being a multitude of villains, for the death of Norman Morbucks (secretly the costumed villain known as the Green Goblin), they were soon defeated and apprehended by the Powerpuff Girls, who had just arrived to save Spider-Man.

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