Sexually active women represent the fastest growing human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we previously reported the synthesis of novel nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase with sperm-immobilizing activity (SIA). To gain further insight into the structure-function relationship controlling these two properties of NNIs, we have rationally designed and synthesized 30 novel thiourea compounds and examined them for dual-function, anti-HIV and spermicidal activity. Twelve of the 30 thiourea compounds exhibited potent anti-HIV activity in the nanomolar range (IC₅₀ = <1–9 nM). Nine of the 30 thiourea derivatives exhibited both anti-HIV and spermicidal activity. Among the phenyl ring-containing thioureas, the 2-fluoro (HI-240) -substituted and 2-chloro (HI-253) -substituted derivatives exhibited potent anti-HIV activity (IC₅₀ = <1 nM) with SIA (EC₅₀ = 70 μM and 147 μM). Among the alicyclic ring-containing thioureas, the 5-bromo (HI-346) and 5-chloro (HI-445) functionalized cyclohexenyl ring-substituted thioureas were the most potent dual-function spermicides (EC₅₀ = 42 and 57 μM), with anti-HIV activity at nanomolar range (IC₅₀ = 3 nM). Unlike nonoxynol-9 (N-9), none of the potent dual-function thiourea compounds were cytotoxic to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations. We conclude that as potent anti-HIV agents with SIA and reduced cytotoxicity when compared with N-9, the phenyl-substituted and cyclohexenyl-substituted thiourea derivatives, especially compounds HI-253 (N-[2-(2-chlorophenethyl)]-N′-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridinyl)]-N′-[2-(1-cyclohexenyl)ethyl-thiourea), and HI-445 (N-[2-(5-chloropyridinyl)]-N′-[2-(1-cyclohexenyl)ethyl-thiourea) show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.