Hepatitis C virus (HCV) is a main cause of chronic liver disease, which may lead to the development of liver cirrhosis and hepatocellular carcinoma. Therapeutic options are still limited in a significant proportion of patients. Small interfering RNAs (siRNAs) are an efficient tool to inhibit gene expression by RNA interference. As HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNA-directed antiviral strategies are likely to successfully block its replication cycle. In this study, a panel of siRNAs was used to target various important regions of the HCV genome [5' untranslated region (UTR), NS3, NS4A, NS4B, NS5B, 3' UTR]. Convergent opposing human H1 and U6 polymerase III promoters were used to generate siRNAs. Target genes in sense and antisense orientation were attached to a luciferase reporter system to test the inhibitory efficiency of both siRNA strands. Our data revealed effective RNA interference against the HCV(+)-strand, the HCV(-)-strand or both strands simultaneously up to 65%. Subsequently, active siRNAs were tested in HCV subgenomic replicon cells and suppression of HCV RNA and NS5B protein levels up to 75% was confirmed. Interestingly, siRNAs that were effective against the sense as well as the antisense strand revealed the greatest inhibitory effects on HCV subgenomic replicons. Additionally, combinations of siRNAs induced a greater inhibition of HCV subgenomic replication of up to 90% proving the potential of this combined antiviral approach.