Abstract: In order to investigate the antibacterial effect and mechanism of total flavonoids from Elephantopus scaber,total flavonoids was extracted from Elephantopus scaber and D101 macroporous resin was applied to purity the crude extract. The antibacterial effect and the minimum inhibitory congcentration(MIC) of total flavonoids from Elephantopus scaber on Escherichia coli and Staphylococcus aureus were analyzed by disk diffusion and double dilution method. The conductivity and scanning electron microscopy were used to analyze the antibacterial mechanism of total flavonoids from Elephantopus scaber on Escherichia coli and Staphylococcus aureus. Results indicated that D101 macroporous resin could purify total flavonoids from Elephantopus scabe and the eluent was deep yellow and clear. At the moment,the concentration of total flavonoids was 0.27 mg/mL.Total flavonoids from Elephantopus scaber significantly inhibited Escherichia coli and Staphylococcus aureus with a MIC value of 0.612 μg/mL. In addition,total flavonoids from Elephantopus scaber made electrical conductivity of Escherichia coli culture solution firstly rose and then slightly fell,but which of Staphylococcus aureus culture solution firstly fell,then slightly rose and finally declined with culture time extension. Total flavonoids from Elephantopus scaber damaged the cytoderm structure and changed the cell membrane permeability. Then the release of intracellular substance affected culture solution electrical conductivity. The morphology of Escherichia coli and Staphylococcus aureus was observed under scanning electron. The total flavonoids from Elephantopus scaber led to the roughness in cell surface and changed the shape of the bacteria. In summary,the growth of the indicator bacteria was inhibited by total flavonoids from Elephantopus scaber.