The anti-hepatitis C virus (HCV) activity of Solanum rantonnetii aerial parts and identity of the bioactive compounds present were evaluated. This led to identification of kaempferol 8-methyl ether (1), quercetin 3-methyl ether (2), kaempferol (3), vitexin (4), isovitexin (5), kaempferol 3-O-alpha-rhamnoside (6), kaempferol 3-O-beta-glucoside (7), and quercetin 3-O-beta-galactoside (8). The results showed that S. rantonnetii extract has a significant anti-HCV activity, with a half-maximal inhibiting concentration of 7 mu g/ml and no apparent toxicity. Furthermore, the extract was shown to inhibit HCV entry, with no effect when added after infection. Compounds (1 and 2) isolated from the extract showed a strong anti-HCV activity and a third compound, compound 3, had a moderate anti-HCV activity. This is the first report for anti-HCV activity of S. rantonnetii and its bioactive compounds. These results suggest that S. rantonnetii extract could become a molecular template for the development of new antiviral therapy for HCV infection.