In present study, we aimed to illustrate activities and actions of flavonoids from stems and leaves of Glycyrrhiza inflata against methicillin-resistant Staphylococcus aureus (MRSA). The different chromatographic methods were firstly used to isolate flavonoids from stems and leaves of G. inflata. Then, the minimum inhibitory concentration (MIC) were determined using the micro-dilution method. Six compounds were obtained and identified as 6-prenylnaringenin, eriodictyol, 8-prenyleriodictyol, 6-prenylnaringenin, isolicoleafol, 6-prenyleriodictyol. The results indicated that 8-isoprenyleriodictyol and 6-isoprenylnaringin (6-PN) exhibited strong inhibitory effects on MRSA, with MIC values of 7.8 µg/mL. The fluorescent probes and multi-omics techniques were used to reveal the antibacterial mechanisms of 6-PN. The fluorescent probe results indicate that 6-PN could cause the membrane damage in MRSA cells. The results of co-incubation with phospholipids indicated that 6-PN may cause MRSA cell membrane damage by interacting with bacterial membrane phospholipids. Furthermore, the metabolomics results indicated that 6-PN could inhibit arginine synthesis in MRSA. The proteomic results indicated that 6-PN could inhibitmet the oxidative stress and arginine of MRSA. Taken together, 6-PN may inhibit MRSA growth though targeting MRSA cell membranes and metabolismarginine synthesis.