Abstract: The aim of this study was to screen strains capable of simultaneously degrading AFB₁ and ZEN from soil samples, fermented food, animal manure, as well as moldy peanuts and corn collected from multiple provinces across the country. The results showed that the strain Bacillus amyloliquefaciens HNGD-Yq12 isolated from Chongqing maize-field soil exhibited degradation capabilities for both AFB₁ and ZEN in its cell-free culture supernatant at 40 °C under neutral pH conditions, with degradation rates of 91.1% and 94.6%, respectively. The temperature-optimization experiments demonstrated that 40 °C was the optimal temperature for the degradation enzyme of this strain. When the temperature deviates from this range, the degradation efficiency is reduced, and, in particular, at temperatures below 30 °C the enzyme activity is markedly diminished, a pattern consistent with the typical activity-temperature characteristics of most microbial enzymes. Mn²⁺ significantly promoted the degradation of both AFB₁ and ZEN, whereas Cu²⁺ specifically enhanced the degradation of AFB₁. The metal-ion influence experiments suggested that different metal ions exert distinct regulatory effects on the degradation process. Notably, Mn²⁺ may serve as a cofactor or activator for certain degradation enzymes, whereas the specific promotion of AFB₁ degradation by Cu²⁺ suggests that the degradation pathways of the two toxins may involve distinct enzymatic systems. Furthermore, based on analysis using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), AFB₁ was transformed into C₁₆H₁₄O₅ and C₁₄H₁₂O₄, whereas ZEN was converted into 13-OH-ZEN-quinone (C₁₈H₂₀O₆) . It is speculated that the toxic structures of these mycotoxins have been disrupted. Mass-spectrometric fragment analysis further revealed that the furan ring and coumarin moiety of AFB₁ may undergo ring-opening reactions, while the lactone ring of ZEN was opened and subsequently oxidized; these structural alterations are identified as the key modifications responsible for the reduction in toxicity. In practical applications, treating peanut powder and corn powder with this strain achieved detoxification rates of 72.18% and 67.29% for AFB₁ and ZEN, respectively. In conclusion, Bacillus amyloliquefaciens HNGD-Yq12 exhibits excellent application potential in the simultaneous degradation of multiple mycotoxins. Future research will focus on the isolation, purification, and identification of degradation-active components, the cloning and heterologous expression of degradation gene clusters, and the development of enzyme preparation processes applicable to large-scale production to facilitate the translation of this technology from laboratory research to practical application.