Abstract: This study aimed to investigate the inhibitory effects of three major flavonoids from the Hippophae rhamnoides subsp. yunnanensis berries on four pathogenic microorganisms. The antimicrobial activity of total flavonoids from Hippophae rhamnoides subsp. yunnanensis berries on Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans was firstly measured by filter paper method. Then, the total flavonoids compositions were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the main components were quantified by high performance liquid chromatography (HPLC). Finally, the Bradford method and the double dilution method were used to assess the antimicrobial effect of the three flavonoids. The results showed that the major flavonoid components of the Hippophae rhamnoides subsp. yunnanensis berries were isorhamnetin-3-O-glucoside-7-O-rhamnoside (Is-3-G-7-Rh), quercetin-3-O-rutinoside (Qu-3-R) and isorhamnetin-3-O-glucoside (Is-3-G), which with the contents of 38.43±0.85, 29.44±0.42 and 21.11±0.55 mg/g, respectively. The extract and its three main flavonoids had inhibitory effect on the four microorganisms. The inhibitory effects of Is-3-G-7-Rh and Is-3-G on the microorganisms were better than Qu-3-R and extract, and the results of protein detection showed that flavonoids induced antimicrobial death through intracellular protein efflux. This study can provide a scientific basis for the application of flavonoids in the berries of Hippophae rhamnoides for antimicrobial purposes.