Abstract: This study aimed to screen bacterial strains capable of simultaneously degrading aflatoxin B₁ (AFB₁) and zearalenone (ZEN) from soil samples, fermented foods, animal manure, as well as moldy peanuts and corn collected from multiple provinces of China. The results showed that Bacillus amyloliquefaciens HNGD-Yq12, isolated from maize-field soil in Chongqing, exhibited dual degradation capacity against AFB₁ and ZEN. Its cell-free culture supernatant achieved degradation rates of 91.1% for AFB₁ and 94.6% for ZEN under neutral pH conditions at 40 ℃. Temperature-optimization experiments confirmed 40 ℃ as the optimal temperature for the strain’s degradation enzymes. Deviation from this temperature significantly reduced the degradation efficiency. In particular, enzyme activity decreased markedly below 30 ℃, which is consistent with the typical temperature-dependent activity characteristics of most microbial enzymes. Mn²⁺ significantly promoted the degradation of both AFB₁ and ZEN, whereas Cu²⁺ specifically enhanced the degradation of AFB₁. Different metal ions exerted distinct regulatory effects on the mycotoxin degradation process. Notably, Mn²⁺ may act as a cofactor or activator for the degradation enzymes, whereas the specific promotion of AFB₁ degradation by Cu²⁺ implies that AFB₁ and ZEN may be degraded via different enzymatic pathways. Furthermore, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis revealed that AFB₁ was transformed into C₁₆H₁₄O₅ and C₁₄H₁₂O₄, whereas ZEN was converted into 13-OH-ZEN-quinone (C₁₈H₂₀O₆). These transformations indicated structural disruption of the toxic moieties of the two mycotoxins. Further mass-spectrometric fragment analysis demonstrated that the furan ring and coumarin structure of AFB₁ underwent ring-opening reactions, and the lactone ring of ZEN was cleaved and oxidized. Such structural modifications are responsible for the reduced toxicity of the degraded products. In practical detoxification applications, the strain treatment achieved detoxification rates of 72.18% for AFB₁ and 67.29% for ZEN in artificially contaminated peanut powder and corn powder, respectively. In conclusion, Bacillus amyloliquefaciens HNGD-Yq12 possesses excellent potential for the simultaneous biodegradation of multiple mycotoxins. Future studies will focus on the isolation, purification, and identification of active degradation components, the cloning and heterologous expression of functional degradation gene clusters, and the development of scalable enzyme preparation processes, to promote the industrial translation and practical application of this detoxification technology.