BackgroundGlycogen metabolism byLactobacillusspp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5–4.5). During bacterial vaginosis (BV), strict and facultative anaerobes includingGardnerella vaginalisbecome predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome.ResultsThe medium simulating vaginal fluid (MSVF) supported growth ofL. jensenii62G,L. gasseri63 AM, andL. crispatusJV-V01, andG. vaginalisJCP8151A at specific initial pH conditions for 30 d.L. jenseniiat all three starting pH levels (pH 4.0, 4.5, and 5.0),G. vaginalisat pH 4.5 and 5.0, andL. gasseriat pH 5.0 exhibited the long-term stationary phase when grown in MSVF.L. gasseriat pH 4.5 andL. crispatusat pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth ofL. jensenii, L. crispatus, andG. vaginalis; onlyL. gasseriwas able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels – pH 4.5 and 5.0 forL. jensenii,L. gasseri, andG. vaginalisand pH 5.0 forL. crispatus.ConclusionsThese results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli andG. vaginalisJCP8151A by affecting their ability to metabolize glycogen. Further, we found thatL. jensenii62G is capable of glycogen metabolism over a broader pH range (4.0–5.0) whileL. crispatusJV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed thatG. vaginalisJCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02916-8.