Staphylococcus aureus is one of a pathogenic bacteria with a high rate of resistance. The discovery of herbal plants that have the potential as an antibacterial is needed. Barleria prionitis is known to have the antibacterial activity of Staphylococcus aureus. The focus of this research is to determine the antibacterial effect of the ethyl hexane fraction of Barleria prionitis on Staphylococcus aureus in mice. This study used an experimental method in which mice treated by injection of Staphylococcus aureus and then given with NaCl (negative control group), Amoxicillin (positive control group) and ethyl hexane fraction Barleria prionitis at a concentration of 0.3 mg / kg; 0.6 mg / kg; 1.2 mg / kg; 233 mg / kg; 4.8 mg / kg (treatment group 1-5). The research found that the Barleria prionitis ethyl hexane fraction with a dose of 2.4 mg/kg and 4.8 mg/kg showed significant bacterial growth inhibition activity. But the ethyl hexane fraction of Barleria prionitis showed no significant antibacterial activity compared to Amoxicillin