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Aflatoxin B₁ (AFB₁) contamination in food and feed is a global health and economic threat, necessitating the immediate development of effective strategies to mitigate its negative effects. This study focuses on the isolation and characterization of <i>Enterococcus faecium</i> HB2-2 (<i>E. faecium</i> HB2-2) as a potent AFB₁-degrading microorganism, using morphological observation, biochemical profiling, and 16S rRNA sequence analysis. An incubation of <i>E. faecium</i> HB2-2 at 32 °C for 96 h in a pH 10 nutrient broth (NB) medium resulted in a remarkable degradation rate of 90.0% for AFB₁. Furthermore, <i>E. faecium</i> HB2-2 demonstrated 82.9% AFB₁ degradation rate in the peanut meal, reducing AFB₁ levels from 105.1 to 17.9 μg/kg. The AFB₁ degradation ability of <i>E. faecium</i> HB2-2 was found to be dependent on the fermentation supernatant. The products of AFB₁ degradation by <i>E. faecium</i> HB2-2 were analyzed by liquid chromatography–mass spectrometry (LC-MS), and a possible degradation mechanism was proposed based on the identified degradation products. Additionally, cytotoxicity assays revealed a significant reduction in the toxicity of the degradation products compared to the parent AFB₁. These findings highlight the potential of <i>E. faecium</i> HB2-2 as a safe and effective method for mitigating AFB₁ contamination in food and feed.