The emergence of HIV/AIDS as a disease spread through sexual intercourse has prompted the search for safe and effective vaginal and rectal microbicides for curbing mucosal viral transmission via semen. Since endogenous reverse transcription is implicated in augmenting the sexual transmission of HIV-1 infection, potential microbicides should have the inherent ability to optimally inhibit both wild-type and drug-escape mutants. The non-nucleoside reverse transcriptase inhibitors (NNRTIs), which bind to an allosteric site on RT, are an important arsenal of drugs against HIV-1. The clinical success of NNRTI-based HIV/AIDS therapies has led to extensive structural and molecular modelling studies of enzyme complexes and chemical synthesis of second- and third-generation NNRTIs. Rationally designed NNRTIs deduced from changes in binding pocket size, shape and residue character that result from clinically observed NNRTI resistance-associated mutations exhibit high binding affinity for HIV-1 RT and robust anti-HIV activity against the wild-type and drug-escape mutants without cytotoxicity. Notably, membrane permeable tight binding NNRTIs have the ability to inactivate cell-free as well as cell-associated HIV-1 in semen without metabolic activation. Consequently, NNRTIs currently under development as experimental microbicides include thiourea-PETT (where PETT stands for phenethylthiazolylthiourea) derivatives (PHI-236, PHI-346 and PHI-443), urea-PETT derivatives (MIV-150), oxypyrimidines (S-DABOs), thiocarboxanilides (UC-781) and diarylpyrimidines (TMC-120). Mucoadhesive formulations of these NNRTIs have been studied for safety and efficacy in animal models and some have entered Phase I safety testing in humans. This review focuses on the structural, biological and preclinical studies relevant to the clinical development of these NNRTIs as molecular virucides intended to prevent the sexual transmission of HIV-1.