Three different polarity extracts were prepared from sunflower stalk pith. The agar diffusion assay was used to investigate the antibacterial activities of the extracts, and the action mechanism of the ethyl acetate extract (EE) against Staphylococcus aureus and Escherichia coli was detected in this study. Results showed that all extracts have good bacteriostatic effects on the four tested bacteria. Both MIC values of Staphylococcus aureus group and Escherichia coli group which treated by EE were 0.78 g/L, and the IC50 valueswere1.11±0.01 and 1.59±0.03g/L, respectively. There was positive correlation between the antibacterial activities and the content of total phenols and total flavonoids (P<0.05).The damaged membrane structures induced by EE were observed by SEM. EE also increased the permeability of the cell wall and cell membrane of the tested bacterial strains with the release of the electrolytes, proteins and alkline phosphatase (AKP) in the cell. Finally, EE also could inhibit the replication of bacterial genetic material (DNA) and ultimately inhibited bacterial reproduction by 4',6-diamidino-2-phenylindole (DAPI) method.