To unlock these prefabs, do the following below.

Prefab	How to unlock
Annie	Successfully complete the Blastlands Bonus Song in Mellow difficulty (or higher)
Bat Gal	Successfully complete the Beat Factory Boss Song in Brutal difficulty (or higher)
Bat Lad	Successfully complete the Metaclouds Bonus Song in Normal difficulty (or higher)
Biggie	Successfully complete the Metaclouds Bonus Song in Mellow difficulty (or higher)
Bot Girl	Successfully complete the Beat Factory Bonus Song in Normal difficulty (or higher)
Bug	Successfully complete the Beat Factory Boss Song in Insane difficulty
Cyclops	Successfully complete the Elektro Kore Bonus Song in Insane difficulty
Demon Dave	Successfully complete the Metaclouds Bonus Song in Brutal difficulty (or higher)
Diabolical D	Successfully complete the Elektro Kore Bonus Song in Brutal difficulty (or higher)
Disco Marv	Successfully complete the Blastlands Bonus Song in Brutal difficulty (or higher)
Evil Motomatic	Successfully complete the Elektro Kore Boss Song in Insane difficulty
Fred	Successfully complete the Neotropolis Bonus Song in Mellow difficulty (or higher)
Goggle Bot	Successfully complete the Beat Factory Bonus Song in Insane difficulty
Grondor	Successfully complete the Beat Factory Boss Song in Normal difficulty (or higher)
Happy Fun Girl	Successfully complete the Beat Factory Boss Song in Mellow difficulty (or higher)
Hotpants	Successfully complete the Metaclouds Boss Song in Normal difficulty (or higher)
Kim	Successfully complete the Blastlands Boss Song in Mellow difficulty (or higher)
Kong	Successfully complete the Beat Factory Bonus Song in Brutal difficulty (or higher)
Larry	Successfully complete the Blastlands Boss Song in Insane difficulty
Lazy Jay	Successfully complete the Elektro Kore Boss Song in Normal difficulty (or higher)
Levar	Successfully complete the Metaclouds Boss Song in Mellow difficulty (or higher)
Little Tony	Successfully complete the Blastlands Boss Song in Brutal difficulty (or higher)
Mad Melvin	Successfully complete the Neotropolis Bonus Song in Insane difficulty
MC Mike	Successfully complete the Blastlands Bonus Song in Normal difficulty (or higher)
Mysterion	Successfully complete the Blastlands Bonus Song in Insane difficulty
Naomi	Successfully complete the Elektro Kore Bonus Song in Mellow difficulty (or higher)
Norm	Successfully complete the Neotropolis Boss Song in Mellow difficulty (or higher)
Pixel	Successfully complete the Elektro Kore Boss Song in Brutal difficulty (or higher)
Rageatron	Successfully complete the Metaclouds Bonus Song in Insane difficulty
Razor X	Successfully complete the Neotropolis Boss Song in Brutal difficulty (or higher)
Robofairy	Successfully complete the Metaclouds Boss Song in Insane difficulty
Roxanne	Successfully complete the Neotropolis Bonus Song in Brutal difficulty (or higher)
Swat Man	Successfully complete the Elektro Kore Bonus Song in Normal difficulty (or higher)
Tex	Successfully complete the Elektro Kore Boss Song in Mellow difficulty (or higher)
Trollheim	Successfully complete the Metaclouds Boss Song in Brutal difficulty (or higher)
Trust Me	Successfully complete the Blastlands Boss Song in Normal difficulty (or higher)
Velma	Successfully complete the Beat Factory Bonus Song in Mellow difficulty (or higher)
Vern	Successfully complete the Neotropolis Boss Song in Normal difficulty (or higher)
Xenon	Successfully complete the Neotropolis Boss Song in Insane difficulty
Zandork 9	Successfully complete the Neotropolis Bonus Song in Normal difficulty (or higher)