The flavonoids in Ageratum conyzoides L. have been used in traditional medicine due to its anti-inflammatory and antibacterial properties. However, the specific mechanism of its antibacterial effect, and the potential therapeutic effect on vaginitis have not been well explained. The growth curves of E. coli, S. aurues, and P. aeruginosa after treatment with flavonoids were measured. The influences of flavonoids on the conductivity of bacterial culture medium and exudation of bacterial nucleic acid were also detected. Transcriptomics analysis was applied to analyze the potential mechanism of flavonoids. Flavonoids significantly suppressed the growth curves of E. coli, S. aurues, and P. aeruginosa, and increased the conductivity of bacteria and nucleic acid exudation. Transcriptomics analysis indicated that flavonoids could suppress bacteria by affecting the transcription and metabolism pathways. The obvious therapeutic effect of flavonoids on bacterial vaginitis was also observed. This study systematically analyzed the bacteriostatic mechanism of flavonoids, which should be helpful to develop new drugs based on the bacteriostatic effect of flavonoids.