Bacterial vaginosis (BV) is a prevalent condition, impacting a third of women globally. It’s marked by reduced healthy lactobacilli and increased growth of certain anaerobes like Gardnerella. Even though antibiotics can be taken orally or applied vaginally to treat BV, about 50% of the patients will have the condition return in less than 6 months. High concentrations of Lactobacillus acidophilus have been linked to health benefits. Given the recurrence rates after treating BV, there’s growing interest in using beneficial bacteria either as an alternative or as a supplementary treatment. This research wanted to demonstrate the potential for a new long-lasting delivery method for L. acidophilus. Results indicated that polyethylene oxide (PEO) fibers containing L. acidophilus can be combined with poly(lactic-co-glycolic acid) (PLGA) fibers in a 1:1 ratio. This structure subsequently releases L. acidophilus, which remains metabolically active, produces lactic acid, and can counteract Gardnerella. When these probiotic-rich fibers were consistently cultured in MRS broth and refreshed daily, they continued to produce active L. acidophilus for up to a week. The amount of lactic acid and associated pH measurements reflected the concentration of L. acidophilus from the fibers, underscoring their metabolic activity. In lab tests with vaginal cell cultures, the L. acidophilus fibers hindered Gardnerella growth in proportion to their dilution, showcasing their bacteria-killing capability. Introducing VK2/E6E7 cells to these L. acidophilus fibers showed only a slight reduction in cell viability compared to cells that weren’t treated. In summary, the findings highlight the potential of using electrospun fibers as a viable means to administer vaginal probiotics in a durable format.