o-Vanillin is a natural product that has been widely applied in the food and pharmaceutical industries. In this study, we determined that o-vanillin can strongly inhibit the growth of Aspergillus flavus mycelia. However, the inhibition mechanism of o-vanillin is still elusive. The ultrastructural morphology of mycelia was injured, and the cell walls were destroyed. The OH functional groups on cell walls were altered, and the content of protein in mycelial cell walls was reduced by o-vanillin. The content of beta-1,3-glucan in cell walls was significantly (P < 0.05) reduced by o-vanillin in a dose-dependent manner, while chitin was not markedly affected. Moreover, o-vanillin led to an increase in the permeability of cell membranes. o-Vanillin also exhibited a promising antifungal effect on contaminated corn kernels. Therefore, o-vanillin inhibited the growth of mycelia by disrupting the integrity of cell walls and cell membranes. This study not only sheds light on the antifungal mechanism of o-vanillin but also indicates that it is a promising agent for the control of A. flavus infection.