In order to determine the chemical composition of Artemisia anethoides Mattf. essential oil, and explore its antibacterial activity and antibacterial mechanism, we extracted the essential oil of Artemisia anethoides Mattf. by the steam distillation, and its chemical constituents were determined by gaschromatographymass spectrum (GCMS) in this study. The antibacterial activity of the essential oil was determined by the inhibition zone method, double dilution method and growth curve method, and the antibacterial mechanism of the essential oil was investigated by the electrical conductivity method and scanning electron microscope method. The results showed that: (1) the main chemical constituents of Artemisia anethoides Mattf. essential oil included alcohols (47.12%) and terpenes (19.90%), among all the components, the content of eucalyptol (12.39%) was the highest, followed by terpineol (8.70%). (2) The diameters of inhibition zone of essential oil against S.aureus and E.coli were (22.57±1.68) mm and (15.36±0.71) mm, respectively. (3) The minimum inhibitory concentrations of essential oil against S. aureus and E. coli were 3.25 and 7.5 μL/mL, respectively, and the minimum bactericidal concentrations were 7.5 and 15 μL/mL, respectively. (4) When the concentration of essential oil were 1.625 and 3.25 μL/mL, it could delay the growth of both S.aureus and E. coli. When the concentrations of essential oil were 3.25 and 7.5 μL/mL, it could inhibit the growth of S. aureus completely, and when the concentrations of essential oil were 7.5 and 15 μL/mL, it could inhibit the growth of E. coli completely. (5) Having been treated with essential oil, the relative conductivity of the bacteria increased significantly, with the increased of the essential oil concentration. In addition, the phenomenon of atrophy and rupture of the cell membrane occurred. The study found that the essential oil of Artemisia anethoides Mattf. was rich in many active substances, such as alcohols and terpenes. It had good antibacterial activity against S.aureus and E.coli, and the essential oil of Artemisia anethoides Mattf. could change the membrane structure of cells and resulted in the leakage of soluble substances in bacteria, thereby inhibit the growth of bacteria.