Paeonol can effectively inhibit Aspergillus flavus (A. flavus) via damaging cell walls. In this work, paeonol treatment remarkably destroyed both the outer amorphous layer and the inner fibrous layer of cell walls. Furthermore, FT-IR and XPS characterization showed that OH functional groups were altered and proteins in the outer layer were released. According to proteomic analysis, 605 proteins have been identified and annotated. The activities of β-1,3-glucan synthase and chitinase were prohibited and promoted, respectively, by paeonol treatment, however, the activities of β-1,3-glucanase and chitin synthase were not influenced. QRT-PCR results suggested that FKSP, CHIIII, and CHIV genes might be the antifungal targets of paeonol. In addition, paeonol can effectively restrain the pathogenicity of A. flavus on peanut butter. This study provided a new elucidation on the mode of action of paeonol against cell walls of A. flavus, facilitating the application of paeonol in the preservation of agricultural products.