Bacterial vaginosis (BV) is a common infectious disease of the lower female reproductive tract, which is characterized by the augmentation of anaerobic bacteria.Gardnerella (G.) vaginalisplays a predominant role in BV recurrence relating to its higher virulence potential and biofilm formation ability. With the increased proportion of metronidazole-resistantG. vaginalis,controlling resistance to metronidazole and finding more effective drugs became a major concern. In this study, 30 clinical strains were cultured from the vaginal secretions of BV patients, followed by PCR and 16S rDNA sequencing identification. According to the CLSI guidelines for anaerobic drug sensitivity testing, 19 strains were identified as metronidazole-resistant (minimum inhibitory concentration, MIC ≥ 32 μg/mL), of which 4 clinical strains were observed to be strong biofilm producer and the final minimum biofilm inhibitory concentration (MBIC) of metronidazole was increased to 512 μg/mL.Sophora flavescensAlkaloids (SFAs), a traditional chinese medicine, could not only inhibit the growth of metronidazole-resistantG. vaginalisin planktonic (MIC: 0.3125–1.25 mg/mL), but also eliminate the biofilm formation (MBIC: 0.625–1.25 mg/mL). In the high-magnification scanning electron, it was observed that the morphology of biofilm changed from a thick to flaky shape and was nearly depleted. These results indicate that SFAs could not only inhibit the growth of metronidazole-resistantG. vaginalisin planktonic and biofilm levels, but also destroyed the biofilm morphology and microstructure, which may contribute to the prevention of BV recurrence.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-023-03378-x.