Previous studies have demonstrated the antibacterial activity of paeonol against bacterial pathogens, but its anti-biofilm activities against Staphylococcus aureus and Listeria monocytogenes remain largely unexplored. Here, the antibacterial and anti-biofilm activities of paeonol against S. aureus and L. monocytogenes were examined using the crystal violet staining assay (CVSA), field emission scanning electron microscopy (FESEM), and confocal laser scanning microscopy (CLSM) analysis. Paeonol effectively inhibited the growth of S. aureus and L. monocytogenes with a minimum inhibitory concentration (MIC) of 500 and 125 μg ml −1 , respectively, and disrupted the integrity of cell membranes. Moreover, sub-MIC paeonol exhibited an inhibitory effect on the attachment of S. aureus and L. monocytogenes to the abiotic surface and biofilm formation. Further, paeonol effectively destroyed cell membranes within biofilms, and dispersed mature biofilms of both strains. The results indicate that paeonol might be a promising antibacterial and anti-biofilm agent for combating infections caused by S. aureus and L. monocytogenes.