Abstract: Mycotoxins are a class of highly toxic metabolites produced by filamentous fungi during their growth and reproduction, posing a significant threat to food security and the health of both humans and animals. In this study, a mixture of zearalenone (ZEN), aflatoxin B₁ (AFB₁), and vomitoxin (DON) was utilized as the sole carbon source to screen for bacteria capable of simultaneously degrading these these three toxins.Bacterial strains were identified through morphological analysis, as well as 16S rRNA and gyrB sequence analysis. By examining the degradation capabilities of bacterial fermentation broth, supernatant, cell suspension, intracellular extract, and inactivated bacterial cells, we aimed to explore the degradation potential of bacterial strains towards three types of toxins and identify the active sites for their degradation. To investigate the degradation properties of the active substances, proteinase K, SDS, and heat treatments were applied to the supernatant. Degradation products were identified using Ultra-High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS). Additionally, optimization of the reaction conditions was conducted by exploring the effects of various metal ions, fermentation temperatures, and pH values to enhance the degradation efficiency of the enzymes. The results indicated that a strain of Bacillus subtilis HNGD-sg5, isolated from sweet garlic, was capable of degrading ZEN, AFB₁, and DON, with degradation rates of 97.27%, 93.81%, and 49.74%, respectively. When all three toxins were present simultaneously, the degradation rates were 99.25%, 98.94%, and 29.86%, respectively. The primary active sites for the degradation of three toxins were located in the fermentation supernatant, where the bacteria exhibited some physical adsorption for ZEN degradation. Further inactivation treatments of the supernatant revealed that the bacteria primarily degraded ZEN, AFB₁, and DON into 15-OH-ZEN, AFB₁-diol, and 3-epi-DON, respectively, through extracellular enzymes. These extracellular enzymes demonstrated high-temperature resistance in the degradation of AFB₁.In the optimization experiment, metal ions Fe³⁺, Ni²⁺, Mn²⁺, and Mg²⁺enhanced the degradation activity of the crude enzyme solution towards ZEN, while Mn²⁺, Cu²⁺, and Zn²⁺enhanced the degradation activity of the crude enzyme solution towards AFB₁. Only Cu²⁺ was found to enhance the degradation of DON. The highest degradation rate of the crude enzyme solution was achieved under fermentation conditions of 50°C and pH 7. These findings indicate that Bacillus subtilis HNGD-sg5 has significant potential for the simultaneous degradation of ZEN, AFB₁, and DON, providing a foundation for the development of fungal toxin-degrading enzyme preparations and the subsequent exploration of related degrading enzyme genes.