The HIV-1 epidemic remains an urgent global health concern. Young women are disproportionately at risk of acquiring the virus. A range of highly effective, female-controlled, discrete vaginal products therefore is needed to help curb the epidemic. Oral tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are effective in HIV-1 pre-exposure prophylaxis (PrEP) and form a promising basis for a vaginal product. Here, we evaluate TDF and FTC in combination with the broadly neutralizing antibody VRC01-N using a highly reproducible humanized mouse model. The agents were vaginally dosed individually and in combination, and the efficacy of HIV-1 prevention was analyzed using the established, rigorous median-effect model. Surprisingly, the triple combination showed a high degree of synergism, unprecedented for in vivo HIV-1 PrEP, leading to a possible fivefold dose reduction for some of the agents. Vaginal administration of the TDF-FTC-VRC01-N combination holds significant promise for HIV-1 PrEP.