Objective To investigate the effects of Honghe Fujie Lotion on Gardnerella vaginalis and its biofilm. Methods A total of 576 patients initially diagnosed with bacterial vaginosis at the Beijing Obstetrics and Gynecology Hospital，Capital Medical University，between November 2022 and June 2023 were enrolled. Ten strains of Gardnerella vaginalis were isolated，purified，and identified from their vaginal secretion samples. Honghe Fujie Lotion was co-cultured with Gardnerella vaginalis for 48 hours or applied to Gardnerella vaginalis pre-cultured for 24 hours. Parameters including OD value，inhibition rate，minimum inhibitory concentration （MIC），and minimum biofilm inhibitory concentration （MBIC） of Gardnerella vaginalis were measured. The status of vaginalis biofilm was observed by scanning electron microscopy. Results The biofilm formation capacity of Gardnerella vaginalis was the strongest after 48 hours of culture （P<0.05）. At the therapeutic dose （10%），the bactericidal rate exceeded 94% within 0-5 minutes，，which showed a rapid，strong and stable bactericidal effect of Honghe Fujie Lotion. Honghe Fujie Lotion could effectively inhibit the growth and biofilm formation of Gardnerella vaginalis even at a low concentration. The inhibitory rates for different strains were different. The MIC value of it ranged from 1/40 to 1/1280 and MBIC value was at 1/80 dilution，still significantly lower than the therapeutic dose. Under the scanning electron microscope，it was found that, even at a concentration lower than the therapeutic dose, Honghe Fujie Lotion could still exert a significant disruptive effect on Gardnerella vaginalis biofilm. Conclusion Honghe Fujie Lotion has a good bactericidal effect on Gardnerella vaginalis and can effectively inhibit the formation of biofilm and destroy the mature biofilm.