The new generation of microbicide candidates is based on the use of antiretroviral (ARV) drugs. The first compounds tested were selected among ARV classes that block the early stages of viral replication cycle, either entry/fusion or reverse transcription. Recently, an additional class of ARVs acting on a later step of viral replication has entered the microbicide pipeline, the protease inhibitors (PIs). This class of ARVs has shown strong potency in highly active antiretroviral therapy (HAART) against human immunodeficiency-1 virus (HIV-1) and lower levels of induced resistance compared to other ARV classes. As candidate microbicides, PIs will have to follow the guiding principles of microbicides being effective, safe, user-friendly and affordable. Hence, aspects including antiviral potency, adverse effects in mucosal tissues, formulation, pharmacology at mucosal sites, emergence of resistance, will have to be considered in the design of a PI-based microbicide. In addition, PIs will have to be tested for their capacity to be used in combination-based microbicides with other ARV classes and in combination with other preventive strategies, such as HIV-1 vaccines. Microbicides containing HIV PIs could also be potentially used against other pathologies specific to the female and/or male genital tract or colorectum.