BACKGROUND: Bacterial vaginosis (BV) is associated with significant reproductive health risks and high recurrence rates after standard antibiotic treatment. Sulfadoxine/pyrimethamine, an antimalarial drug, demonstrated unexpected clearance of BV in clinical trials, suggesting potential antimicrobial effects. Drug repurposing, which leverages existing drugs for new therapeutic applications, offers a promising approach to address the challenges of antimicrobial resistance and high recurrence rates in BV. OBJECTIVE: To determine the in vitro activity of sulfadoxine/pyrimethamine and its components, sulfadoxine and pyrimethamine, on key species associated with BV. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined for sulfadoxine/pyrimethamine (20:1 ratio), sulfadoxine, pyrimethamine, and standard-of-care antibiotics, metronidazole and clindamycin, against BV-associated species (Gardnerella spp., Fannyhessea vaginae, Prevotella bivia) and Lactobacillus crispatus (vaginal health marker). Gardnerella biofilms were also exposed to sulfadoxine/pyrimethamine, pyrimethamine, or metronidazole, and biofilm biomass and biofilm cells culturability were assessed. RESULTS: Sulfadoxine had no effect, while pyrimethamine inhibited all Gardnerella strains with MIC values ranging from 0.125 to 4 mg/L, lower than MICs observed for metronidazole (2-128 mg/L). Pyrimethamine also outperformed metronidazole in inhibiting biofilm mass accumulation and reducing biofilm culturable cells in 3/4 Gardnerella strains. Sulfadoxine/pyrimethamine presented lower MICs than metronidazole for 5/8 Gardnerella strains. Sulfadoxine, pyrimethamine, and sulfadoxine/pyrimethamine showed no activity against other BV-associated species or L. crispatus. CONCLUSIONS: These findings suggest that pyrimethamine (and sulfadoxine/pyrimethamine) could be promising alternative or adjuvant therapies for BV, warranting further clinical trials.