Bacterial vaginosis (BV) is the most common vaginal infection worldwide. We developed a peptide nucleic acid (PNA) probe targetingPrevotella bivia, a common BV-associated bacteria, and optimized a multiplex approach for detection ofGardnerellaspp.,P. biviaandFannyhessea vaginae. OurP. biviaPNA probe specifically detected the target species, and the optimized multiplex approach was able to detect the presence of the three species in multi-species BV biofilms.