Abstract: Aspergillus flavus (A. flavus) and its secondary metabolite aflatoxin (AFT) pose a serious threat to human health and food safety. Therefore, studying strategies for preventing and controlling their contamination is crucial for ensuring food safety and public health. Recently, a plant-derived natural compound, zingerone, has been screened out and found to have a good antibacterial effect against A. flavus. The effect of zingerone on the cell wall and cell membrane of A. flavus were determined by measuring the dry weight of mycelium, observing the integrity of the mycelial cell wall and cell membrane, and measuring the relative conductivity (RC), pH value, total lipid content, and malondialdehyde (MDA) content. Finally, its antibacterial effect in maize was evaluated. The results indicated that the minimal inhibitory concentration (MIC) of zingerone against the growth of A. flavus was 250 μg/mL. The treatments were administered using zingerone at concentrations of 0, 1/4 MIC, 1/2 MIC, and MIC to A. flavus. Compared with the control group, the results showed that the dry weight of mycelium in the treatment group was significantly reduced. Fluorescent staining experiments using Calcofluor White (CW) and propidium iodide (PI) demonstrated that the integrity of the cell wall and cell membrane was significantly disrupted. As indicated by increased extracellular RC, decreased pH, and reduced total lipid and MDA content, suggesting lipid peroxidation and oxidative damage. Notably, zingerone at the MIC completely inhibited A. flavus growth on maize kernels. These findings demonstrate that zingerone exerts its antifungal effects by targeting cell wall and membrane integrity, inducing oxidative stress, and disrupting cellular metabolism. This study provides a solid theoretical foundation and technical support for effectively dealing with A. flavus contamination and offers reference value for the prevention and control strategies in the field of grain storage and food safety.