This study investigates the degradation of resistant α-1,4-glucan by vaginal bacterial species, with a focus on Gardnerella spp., to elucidate its role in bacterial vaginosis (BV). The ability of Gardnerella vaginalis, Gardnerella swidsinskii, Gardnerella leopoldii, Gardnerella piotii, Lactobacillus iners, and Lactobacillus crispatus was assessed to metabolize an ungelatinized, labeled form of raw amylose, a degradation-resistant α-1,4-glucan. The enzymatic activity of these species was evaluated in vitro, and its association with BV was examined in vaginal swabs. Gardnerella vaginalis, G. swidsinskii, and G. leopoldii demonstrated the best ability to degrade resistant α-1,4-glucan in vitro. Unlike the cell-bound, S-layer-associated glycogen-degrading activity in L. crispatus, this α-glucosidase activity in Gardnerella was also extracellular, but not cell-bound and not repressed by glucose. Vaginal swabs showing high rates of resistant α-1,4-glucan degradation activity were associated with BV, particularly in the concurrent presence of G. leopoldii, G. swidsinskii, and G. vaginalis. These findings suggest a role of α-1,4-glucan degradation in BV pathogenesis mediated by Gardnerella species. The results indicate the potential of targeting bacterial amylase activity as therapeutic strategy for BV prevention and treatment.