Background: Mobiluncus spp are highly associated with bacterial vaginosis, but their role in its pathogenesis is unknown. The authors used polymerase chain reaction (PCR) to compare the prevalence of Mobiluncus in women with and without bacterial vaginosis. Goal: To compare the prevalence of Mobiluncus spp among women with and without bacterial vaginosis and to compare the sensitivities of PCR and Gram stain for detection. Study Design: Vaginal specimens from 74 women were analyzed by PCR and Gram stain for the presence of Mobiluncus spp. Comparisons were made between the prevalence of this organism between the two cohorts and between the Gram stain and PCR detection methods. Results: Mobiluncus was detected by PCR in 84.5% of women with bacterial vaginosis and in 38% of women without infection. M curtisii was rarely detected in the latter group, though it was found in 65.3% of women with bacterial vaginosis. The sensitivity and specificity of Gram stain compared with PCR were 46.9% and 100%, respectively. Conclusions: Mobiluncus is more common in healthy women than previously suspected, with M mulieris as the predominant species. The significant difference in the prevalence of M curtisii between women with bacterial vaginosis and uninfected women suggests that this species could be involved in the pathogenesis of bacterial vaginosis.