The development of prevention strategies against sexual transmission of human immunodeficiency-1 virus (HIV-1) is essential to curb the rate of new infections. New prevention options include microbicides, many of which are based on antiretroviral (ARV) drugs targeting different stages of the viral replication cycle including: viral entry and fusion; reverse transcription; integration; and viral maturation through proteolytic clevage. In this review, we discuss current and new potential candidate microbicides designed to prevent mucosal HIV acquisition. Preclinical methods, including cellular, tissular, and animal models, used to assess candidate microbicides and evaluate their prioritization for progress through the product development pipeline are reviewed in context with a rapidly evolving clinical landscape.