Aspergillus flavus contamination has long been a major problem in the food and agriculture industries, while peppermint essential oil (PEO) is increasingly recognized as an effective alternative for controlling fungal spoilage. However, its biocontrol effect and action mode on A. flavus have rarely been reported. Here, the inhibition rates of PEO on A. flavus were determined by the plate fumigation and mycelial dry weight method. The minimum inhibitory concentration (MIC) was identified as 0.343 μL/mL. In the biocontrol tests, the moldy rates of maize kernels, wheat grains, and peanut kernels in the PEO treatment group were significantly reduced by 65%, 72%, and 63.33%, respectively. The biocontrol efficacy of PEO on maize kernels, wheat grains, and peanut kernels reached 80.67%, 82%, and 67.67%, respectively. Furthermore, antifungal action mode analysis showed that PEO changed the mycelial morphology, damaged the integrity of cell wall and membrane. Moreover, it reduced the ergosterol content, elevated the malondialdehyde content, increased the relative conductivity, and led to the intracellular leakage of nucleic acids and proteins, thereby enhancing the cell membrane permeability. In addition, PEO decreased the antioxidant-related catalase (CAT) and superoxide dismutase (SOD) activities, significantly increased the hydrogen peroxide (H