Current treatment options for bacterial vaginosis (BV) have been shown to be inadequate at preventing recurrence and do not provide protection against associated infections, such as that with HIV. This study examines the feasibility of incorporating the antimicrobial peptide subtilosin within covalently cross-linked polyethylene glycol (PEG)-based hydrogels for vaginal administration. The PEG-based hydrogels (4% and 6% [wt/vol]) provided a two-phase release of subtilosin, with an initial rapid release rate of 4.0 μg/h (0 to 12 h) followed by a slow, sustained release rate of 0.26 μg/h (12 to 120 h). The subtilosin-containing hydrogels inhibited the growth of the major BV-associated pathogen Gardnerella vaginalis with a reduction of 8 log10 CFU/ml with hydrogels containing ≥15 μg entrapped subtilosin. In addition, the growth of four common species of vaginal lactobacilli was not significantly inhibited in the presence of the subtilosin-containing hydrogels. The above findings demonstrate the potential application of vaginal subtilosin-containing hydrogels for prophylaxis of BV.