Objective:To investigate the effect of Orostachys (O.) japonicus,a perennial herbaceous plant of the Family Crassulaceae,on biofilm formed by methicillin-resistant Staphylococcus aureus (MRSA).Methods:Powdered O.japonicus was extracted by 95％ methanol,concentrated,and then,systematically fractionated with n-hexane,dichloromethane (DCM),ethyl acetate (EtOAc),n-butanol,and H2O according to polarity.Among them,the flavonoid-rich EtOAc fraction demonstrated the highest antibacterial activity and was used in this study.Using the biofilm inhibition assay,cell-surface attachment assay,confocal laser scanning microscopy,latex agglutination assay,and real time qRT-PCR,we examined whether the EtOAc fraction inhibited the formation of MRSA biofilm.Results:The EtOAc fraction exhibited distinct activity against biofilm formation and cell-surface attachment of MRSA up to 1 mg/mL through down-regulating the expression of mecA gene and the production and agglutination of penicillin-binding protein 2a as solidly observed in biofilm inhibition assay,cell-suface attachment assay,confocal laser scanning microscopy,latex agglutination assay,and real time qRT-PCR analysis.Conclusions:These results suggest that O.japonicus could be utilized as a potential resource for the development of new antibiofilm formation of MRSA and antibacterial agents in the future.