Vestibulodynia is vulvodynia localized to the vulvar vestibule and is a chronic disease defined as vulvar pain of at least three months’ duration, without a defined cause, that severely affects women’s health and quality of life with limited treatment options. We collected stool and vaginal samples from 30 women affected by vestibulodynia and 27 healthy women. Bacterial gut and vaginal microbiomes were characterized by amplicon sequencing, and compositional and functional differences between the control and the patient groups were assessed. No differences in vaginal or fecal alpha and beta diversity were found, but vaginal microbiota of patients was found to be associated with Lactobacillus iners. Moreover, the relative abundance of L. iners negatively correlated with the relative abundance of L. crispatus, and positive correlations between commensals and pathobionts were found in the vestibulodynia vaginal microbiota but not in the healthy controls. The bacterial functions and contributors were defined in the study groups for the fecal and vaginal microbiota. Our results portrayed the vaginal microbiome of patients with vestibulodynia as potentially not as efficient at living in an anaerobic environment as the healthy microbiome is and too inclined to acidify this environment, exposing it to the risk of developing other ailments.