We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.