We and others have shown nonstructural protein 5A (NS5A) to augment hepatitis C virus (HCV) internal ribosomal entry site (IRES)-mediated translation. We have also co-immunoprecipitated heat shock protein (HSP) 70 with NS5A and demonstrated cellular colocalization leading to the hypothesis that NS5A/HSP70 complex formation is important for IRES-mediated translation. Further, we have shown that the bioflavonoid quercetin, an HSP synthesis inhibitor, and HSP70 knockdown block NS5A-augmentated IRES-mediated translation and infectious virus production. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation and NS5A-augmented IRES-mediated translation, whereas catechin and naringenin demonstrated mild activity. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. We also identified the NS5A region binding to HSP70 through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) to be the interaction site. C34 expression significantly reduced intracellular viral protein levels and NS5A-augmented IRES-mediated translation, in contrast to same size control peptides from other NS5A domains. Triple-alanine scan mutagenesis identified an exposed beta-sheet hairpin in C34 to be primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10 amino acid peptide derivative of C34 hairpin suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. We also identified four conserved amino acids in C34 hairpin and showed that altering three of these residues can modulate antiviral activity of the peptides. These results demonstrate the antiviral activity of a number of bioflavonoids, support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, and identify a sequence-specific hairpin element in NS5A responsible for complex formation. The peptides corresponding to this hairpin as well as quercetin, catechin, and naringenin are candidates for HCV therapy that target cellular machinery rather than viral proteins reducing the likelihood of viral resistance.