Abstract Dysbiosis of the vaginal microbiome poses a serious risk for sexual HIV-1 transmission. Prevotella spp. are abundant during vaginal dysbiosis and associated with enhanced HIV-1 susceptibility; however, underlying mechanisms remain unclear. Here, we investigated the direct effect of vaginal bacteria on HIV-1 susceptibility of vaginal CD4+ T cells. Notably, pre-exposure to Prevotella timonensis enhanced HIV-1 uptake by vaginal T cells, leading to increased viral fusion and enhanced virus production. Pre-exposure to antiretroviral inhibitors abolished Prevotella timonensis-enhanced infection. Hence, our study shows that the vaginal microbiome directly affects mucosal CD4+ T cell susceptibility, emphasising importance of vaginal dysbiosis diagnosis and treatment.