Abstract Background Bacterial vaginosis (BV), the most common cause of vaginal discharge, is characterized by the presence of a polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp., but also other anaerobic species. Interactions between bacteria in multi-species biofilms are likely to contribute to increased virulence and to enhanced antimicrobial tolerance observed in vivo. However, functional studies addressing this question are lacking. Objectives To gain insights into the role that interactions between BV-associated species in multi-species BV biofilms might have on antimicrobial tolerance, single- and triple-species biofilms formed by Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae and Peptostreptococcus anaerobius were characterized, before and after metronidazole or clindamycin treatment. Methods Total biofilm biomass, total cells and cfu counts prior to and after antibiotic treatment were first determined. In addition, bacterial populations in the triple-species biofilms were also quantified by quantitative PCR (qPCR) and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Results Despite the effect observed in single-species biofilms, neither metronidazole nor clindamycin was effective in reducing triple-species biofilm biomass. Similar results were obtained when evaluating the number of total or culturable cells. Interestingly, despite differences between strain susceptibilities to antibiotics, the composition of the triple-species biofilms was not strongly affected by antibiotics. Conclusions Taken together, these results strengthen the idea that, when co-incubated, bacteria can interact synergistically, leading to increased tolerance to antimicrobial therapy, which helps explain the observed clinically high BV recurrence rates.