Despite recent advances in curing chronic hepatitis C (CHC), the high economic burden to therapy, viral drug resistance, difficult to treat hepatitis C virus (HCV) genotypes and patient groups are still of concern. To address this unmet medical needs, we devised strategies to identify novel viral interventions through target-free high-throughput screening of small molecules utilizing a phenotypic-based HCV infection assay. Thereby, a very potent (EC50 46 ± 26 pM) iminodipyridinopyrimidine (IDPP) drug candidate was selected, and confirmed in primary human hepatocytes (EC50 0.5 nM). IDPP mainly targets a post-attachment step of HCV without affecting endosomal acidification, prevents the secretion of infectious particles and viral cell-to-cell spread. The putative molecular target of IDPP is glycoprotein E1, as revealed by selection for viral drug resistance (Gly-257-Arg). IDPP was synergistic in combination with FDA-approved HCV drugs and inhibited pre-existing resistant HCV strains induced by today's therapies. Interestingly, IDPP exclusively inhibited HCV genotype 2. However, we identified the genotype-specificity determining region in E1 and generated HCV genotype 1 susceptible to IDPP by changing one amino acid in E1 (Gln-257-Gly). Together, our results indicate an opportunity to provide an alternative treatment option for CHC and will shed light on the poorly understood function of HCV glycoprotein E1.