Hyperosmolar vaginal lubricants are frequently used during sex and in clinical procedures like transvaginal ultrasound (TVUS), but in vitro data suggest they may disrupt the vaginal epithelium and microbiota which play an important role in reproductive tract health. A vaginal microbiota which is dominated by Lactobacillus is associated with optimal reproductive tract health in premenopausal women and fewer symptoms of genitourinary syndrome of menopause among postmenopausal women, whereas a low-Lactobacillus microbiota is associated with bacterial vaginosis (BV) and increased risk for sexually transmitted infections. There is limited data on the effect of hyperosmolar lubricants on vaginal health in vivo from either clinical trials or observational studies. Additionally, no studies have assessed lubricant in a standardized manner controlling for product type, osmolality, ingredients, and dose including frequency and quantity. We utilized a cohort of women presenting for TVUS to evaluate the effect of a single exposure to hyperosmolar lubricant during TVUS on the vaginal microbiota and BV over 10 weeks. We found some limited evidence that a single exposure to hyperosmolar lubricant may be associated with changes in the vaginal microbiota or BV within approximately one week. However, analyses using the full 10 weeks of follow-up indicated peri/postmenopausal participants and participants with a prior history of BV were more likely to have a low-Lactobacillus vaginal microbiota in samples taken twice-weekly for nine weeks after TVUS compared to daily samples collected the week prior to TVUS. Furthermore, an analysis comparing reproductive-age participants in the TVUS cohort with a similar cohort of reproductive-age women without recent lubricant use found that Black lubricant-exposed participants were twice as likely as Black lubricant-unexposed participants to develop incident BV within 10 weeks. Overall, this research demonstrates that a single use of hyperosmolar lubricant may disturb the vaginal microbiota in some populations with a potentially less resilient vaginal microbiota such as women who are peri/postmenopausal, Black, or with a history of BV. Future studies could explore if other populations may be similarly affected given more frequent lubricant use and whether iso-osmolar lubricants have different effects than hyperosmolar lubricants.