Background: Menstrual cups are reusable flexible collectors adjustable to the body. Besides the economic and environmental advantages, it is possible that the use of menstrual cups influences modifications in the vaginal microbiota. We aimed to evaluate the influence of menstrual cups on the vaginal microbiota of reproductive-age women in comparison to sanitary pads and to determine its acceptance. Methods: A prospective longitudinal study was conducted with undergraduate/graduate volunteers, whose usual method of menstrual management was sanitary pads. In the first three menstrual cycles (M1 to M3), participants kept using sanitary pads. In the three consecutive cycles (M4 to M6), they used the menstrual cup offered by the research group. A questionnaire was used to obtain gynecological background and sexual behavior. In the first (M1) and third (M3) follow-up visits, participants underwent gynecological examinations including vaginal microbiota evaluation, oncotic cytology, diagnosis of Human Papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae, and Trichomonas vaginalis. At each visit, vaginal samples were collected for analysis of microbiota. A generalized linear model with binomial distribution for repeated measures was used and the model was adjusted for confounding factors, using the SAS software. Results and Discussion: We included 47 participants who completed the 6 follow-up examinations. The prevalence of HPV was 48.9% at M1 and 51.1% at M3 and the prevalence of C. trachomatis infection was 2.1% and 6.7%, respectively. None of the participants were infected by N. gonorrhoeae or T. vaginalis. One participant had altered cytology results and was referred to the colposcopy service. Patients with C. trachomatis and/or altered vaginal microbiota were treated. During the three months of sanitary pad usage, 27.7%, 12.8%, and 10.6% of women presented altered vaginal microbiota (bacterial vaginosis, intermediate microbiota, aerobic vaginitis, and/or vulvovaginal candidiasis), while only 8.5%, 12.8% and 8.5% of them had vaginal dysbiosis while using a menstrual cup (P = 0.04). Overall acceptance of menstrual cups among those who completed all the examinations was 93.6%. Conclusion: Our results show that menstrual cups are associated with the maintenance of healthy vaginal microbiota. Additionally, the acceptance of menstrual cups was high in our population.