Abstract: Aspergillus flavus (A. flavus) and its secondary metabolite, aflatoxin (AFT), seriously damage the economic value of food, endanger food safety and the health of humans and livestock. Therefore, it is urgent to control the contamination of A. flavus. Our previous results have proved that o-vanillin exerts high efficacy in inhibiting the growth of A. flavus, and the primary mode of action has been determined. This study further figured out more antifungal actions of o-vanillin against A. flavus on the aspects of spore germination, respiration changes, and damage of mycelial cell membrane at different time points (0, 4, 8 and 12 h). At last, the antifungal effects of o-vanillin on the pathogenicity of A. flavus on rice and sunflower seeds were also evaluated. The minimum inhibitory concentration of o-vanillin is 100 μg/mL, which has been determined as the MIC previously in our lab. Gradient concentrations of o-vanillin used in all experiments were 0 (set as control), 25, 50, and 100 μg/mL. The results showed that after 6 h, spores not treated with o-vanillin showed increased volume and early germination was observed. For those treated with o-vanillin at 25, 50, and 100 μg/mL, their size was small and they were still floating, no germinated spores were observed. Hence, the morphology of early germination has been visualized and the effect of o-vanillin within this period of time has been confirmed. Moreover, we tested the dissolved oxygen consumed by spores, but there were no significant changes within 12 hours after treatment of o-vanillin even at MIC. PI staining directly suggested that o-vanillin can also damage the cell membrane of A. flavus in a dose-dependent manner. In detail, the fluorescence intensities of 25, 50, and 100 μg/mL treated mycelia in 12 h were 5.01, 9.42, and 20.00 fold in comparison with that of the control (set as 1). Besides, the integrity of the cell membrane was indicated by cell nucleic acid release, relative conductivity and pH values. The changes of the three indicators were significant and proportional to the treatment time and concentrations of o-vanillin. Finally, o-vanillin can effectively control the pathogenicity of A. flavus on rice and sunflower seeds in 72 h, where complete inhibition was observed on 100 μg/mL of o-vanillin treated samples. In summary, this study suggests that o-vanillin can be developed as a potential antifungal agent against A. flavus, and provides a theoretical basis for the application of o-vanillin in the preservation of grains and agricultural products.