Zearalenone (ZEN) is an estrogenic mycotoxin which can cause loss in animal production. The aim of this study was to screen Bacillus strains for their ZEN detoxification capability and use a fermentation process to validate their potential application in the feed industry. In the high-level ZEN-contaminated maize (5 mg·kg−1) fermentation test, B2 strain exhibited the highest detoxification rate, removing 56% of the ZEN. However, B2 strain was not the strain with the highest ZEN detoxification in the culturing media. When B2 grew in TSB medium with ZEN, it had higher bacterial numbers, lactic acid, acetic acid, total volatile fatty acids, and ammonia nitrogen. The ZEN-contaminated maize fermented by B2 strain had better fermentation characteristics (lactic acid > 110 mmol·L−1; acetic acid < 20 mmol·L−1; pH < 4.5) than ZEN-free maize. Furthermore, B2 also had detoxification capabilities toward aflatoxins B1, deoxynivalenol, fumonisin B1, and T2 toxin. Our study demonstrated differences in screening outcome between bacterial culturing conditions and the maize fermentation process. This is important for the feed industry to consider when choosing a proper method to screen candidate isolates for the pretreatment of ZEN-contaminated maize. It appears that using the fermentation process to address the ZEN-contaminated maize problem in animal feed is a reliable choice.