Oregano essential oil as long been known for its health promoting benefits. Here we report its activity against viral replication. Oregano oil was found to specifically inhibit lentiviruses such as Human and Simian Immunodeficiency Viruses (HIV and SIV), irrespective of virus tropism, but not Hepatitis C, Adenovirus (ADV5), ZIKA and Influenza (H1N1) viruses. Oregano oil's most abundant components, carvacrol and its isomer thymol, were shown to block virus-target cell fusion, while not perturbing other stages of the viral life cycle. We detected changes in viral particle density suggesting that cholesterol depletion from the HIV-1 envelope membrane reduces viral entry. Furthermore, infection was rescued by adding exogenous cholesterol. Evolution of viral resistance to carvacrol supported this mechanism of action with the identification of mutations in the viral gp41 fusion protein that counteracted cholesterol depletion. In addition, resistance to carvacrol emerged later than typically observed for other clinically used drugs, strengthening its antiviral potential. Structure activity relationship studies revealed key motifs of carvacrol and thymol required for HIV neutralization and identified previously unknown active analogs. Carvacrol was also shown to additively cooperate with antiretroviral therapy. In sum, oregano oil and improved carvacrol and thymol analogs could be considered to supplement current HIV therapeutics.IMPORTANCE Oregano essential oil has multiple benefits in traditional medicine, cosmetic and food industries. Carvacrol and its analog thymol are well-described components of oregano oil. Here we show that these compounds inhibit HIV-target cell fusion, independently of viral tropism. Our results suggest that carvacrol and thymol alter the cholesterol content of the viral membrane, blocking HIV-1 entry into the target cell. Resistance to carvacrol has selected for viruses with mutations in the viral envelope glycoprotein, gp41. This protein is known for its interaction with cholesterol present in membrane lipid rafts. Together these results demonstrate the potential of therapies targeting the viral envelope membrane and oregano oil has a safe supplement to antiretrovirals, potentially delaying disease progression and resistance development.