Hemolysis test, coagulase titer assay, and enzyme-linked immunoreaction method were used to detect the expression changes of hemolysin, plasma coagulase, and total enterotoxins after treating with punicalagin. Reverse transcription real-time fluorescence quantitative PCR was used to detect the expression of virulence-related genes. The results showed that punicalagin could significantly reduce the production of α-hemolysin, but the inhibition effect was not concentration-dependent. At the concentrations of higher than 1/4 MIC, punicalagin could decrease the expression of plasma coagulase. At the concentrations of higher than 1/2 MIC, punicalagin significantly reduced the production of total enterotoxins. Punicalagin also significantly reduced the transcription of enterotoxin gene sea, hemolysin gene hla, and regulatory gene agrA. The relative expression levels of the three genes were 3%, 5% and 5% of the control group at 1/8 MIC of punicalagin, respectively.