Abstract: OBJECTIVE To explore the in vitro antibacterial activity and action mechanisms of the quinones compounds thymoquinone,extracted from natural fennelflower seeds,against methicillin resistant Staphylococcus aureus (MRSA) so as to provide theoretical bases for development of novel anti MRSA drugs. METHODS The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thymoquinone against MRSA standard strains were determined by using the microdilution broth method and agar plate spread method,respectively. The antimicrobial kinetics were evaluated by plotting growth curves. The interventional effects of thymoquinone on biofilm formation and mature biofilms were evaluated by crystal violet staining. The changes in bacterial ultrastructure were observed by using scanning/transmission electron microscope. The intracellular reactive oxygen species (ROS) levels were detected by means of the DCFH-DA fluorescent probe. RESULTS Booth the MIC and MBC of thymoquinone against MRSA were 16 μg/ml. The growth curves demonstrated that its antibacterial effect presented with concentration dependent characteristic, it has remarkable antibacterial effect at the concentration of 16 μg/ml and completely inhibited bacterial growth at 64 μg/ml. Thymoquinone could effectively inhibit the biofilm formation and clear the established mature biofilms. Thymoquinone could disrupt the cell membrane integrity, leading to leakage of contents and bacterial lysis. Simultaneously, it could induce the substantial accumulation of ROS within bacteria and exacerbate oxidative damage. CONCLUSIONS Thymoquinone exhibits significant bactericidal activity against MRSA and effectively interferes with biofilm formation. The action mechanisms are closely associated with its disrupting cell membrane structure and inducing oxidative stress.