HIV-1 transmission occurs mostly through sexual contact with infected individual. HIV crosses vaginal mucosa and HIV-1 can infect dendritic cells, macrophages and T lymphocytes in the submucosa and then target circulating CD4+ T cells. In the developing world, women have limited freedom to choose sexual situations or to insist on condom use. In such scenario, microbicide would prove an effective prevention tool. Therefore, the development of an anti-HIV microbicide is extremely important. The recent trend in the development of new microbicide candidates includes the utilization of FDA-approved drugs for HIV therapy that target the early stages of the HIV life cycle, including entry inhibitors and reverse transcriptase (RT) inhibitors. We have evaluated 4-BPMDP, a potent reverse transcriptase inhibitor, for its spectrum of anti-HIV-1 activity in hPBMCs. 4-BPMDP significantly inhibited several strains of HIV-1 including X4, R5 and X4R5 dual tropic viruses. It also inhibited replication of subtype C R5 tropic clinical isolates at low micromolar concentrations. Further we investigated 4-BPMDP as microbicide candidate. 4-BPMDP is nontoxic to vaginal epithelial cells as evident from cytokine analysis. In dual chamber model system of cervical epithelial cells, 4-BPMDP moderately inhibited transmission of HIV-1NL4-3. Our data indicate that 4-BPMDP displays potent anti-HIV-1 activity and could serve as tools for the identification of novel anti-HIV targets. However further chemical modification would be needed to enhance its HIV-1 microbicidal properties.