Abstract: OBJECTIVE To explore the antibacterial activity of prenylated flavonoids from Sophora flavecens and licorice flavonoids and their combination in vitro and in vivo， so as to provide scientific basis for the resource value discovery and comprehensive development of Sophora flavescens Ait. and Glycyrrhiza uralensis Fisch. METHODS The UPLC-Q-TOF-MS technology was applied to qualitatively analyze the prepared prenylated flavonoids from Sophora flavescens Ait. (KSHT) and licorice flavonoids (GCHT). The in vitro antibacterial activity was evaluated by investigating the effect of a single use and combined use of KSHT and GCHT on inhibiting the growth of Staphylococcus aureus and the generation of biofilm and flavin. The in vivo antibacterial activity was evaluated by building a mouse mastitis model induced by Staphylococcus aureus. RESULTS A total of 20 and 22 flavonoids were identified from KSHT and GCHT， respectively. KSHT outperformed GCHT in inhibiting the growth of Staphylococcus aureus and the generation of biofilm and flavin. Moreover， the combined use of KSHT and GCHT had better inhibitory effect than their independent use， suggesting that they have synergistic antibacterial action. Whether they were independently or jointly used in high or low doses， all the indexes of mastitis in mice were improved. Compared with the independent use of KSHT and GCHT， the combined use can better improve the viscera index and fungal burden of tissue in mice with mastitis. Regarding the improvement of indexes， KSHT was better than GCHT. In terms of the effect on improving the histopathology of mammary gland and reducing IL-1β， IL-2 and TNF-α in model group， GCHT was better than KSHT， but equivalent to the combination group. CONCLUSION The results of in vitro and in vivo tests indicated that the combined use of KSHT and GCHT has the best effect in resisting Staphylococcus aureus. It is expected that they can be jointly used in the prevention and treatment of mastitis in animal husbandry.