In this work, the antibacterial activity and mechanism of chloroform fraction obtained from aqueous extract of mugwort leaves against Staphylococcus aureus were investigated. The extract showed obvious antibacterial activity against S. aureus which the minimum inhibitory concentration and minimum bactericidal concentration were determined to be 3·0 and 6·0 mg ml−1 respectively. The mechanism study suggested that the extract could destroy the integrity of the S. aureus cell walls and increase the permeability of cell membrane in a certain concentration, but it could not kill S. aureus in a short time. Instead, the extract could make bacteria in a state of apoptosis for a long time, interfere with the normal physiological metabolism of bacteria, and eventually make bacteria die, which was confirm by scanning electronic microscope. Significance and Impact of the Study: Since ancient times, Mugwort (Artemisia argyi L.) has been widely used as a traditional medicine for many diseases including bacterial infectious diseases; therefore, it plays a very important role in protecting people’s health. Previous studies have proved that the essential oil of mugwort has antibacterial activity; however, the antibacterial activity of the non‐volatile components of it is still unknown. In this work, the antibacterial activity and mechanism of aqueous extract of mugwort leaves were investigated for the first time. The results obtained from this study provide a scientific basis for revealing the antibacterial activity of the non‐volatile components of mugwort, and lays a foundation for further development and utilization of mugwort.