In previous studies, authors have completed the total synthesis of several phloroglucinol natural products and synthesized a series of their derivatives, which were tested with good biological activities.To discover anti-MRSA lead compound and study their mechanism of action.Phloroglucinol derivatives were tested to investigate their activities against several gram-positive strains including Methicillin-resistant Staphylococcus aureus (MRSA). The mechanism study was conducted by determining extracellular potassium ion concentration, intracellular NADPH oxidase content, SOD activity, ROS amount in MRSA and MRSA survival rate under A5 treatment. The in vitro cytotoxicity test of A5 was conducted.The activity of monocyclic compounds was stronger than that of bicyclic compounds, and compound A5 showed the best MIC value of 0.98 μg/mL and MBC value of 1.95 μg/mL, which were 4-8 times lower than that of vancomycin. The mechanism study of A5 showed that it achieved anti-MRSA effect through membrane damage, which is proved by increased concentration of extracellular potassium ion after A5 treatment. Another possible mechanism is the over ROS production induced cell death, which is suggested by observed alternation of several reactive oxygen species (ROS) related indicators including NADPH concentration, superoxide dismutase (SOD) activity, ROS content and bacterial survival rate after A5 treatment. The cytotoxicity results in vitro showed that A5 was basically non-toxic to cells.Acylphloroglucinol derivative A5 showed good anti-MRSA activity, possibly via membrane damage and ROS-mediated oxidative stress mechanism. It deserves further exploration to be a potential lead for the development of new anti-MRSA agent.