A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of ∼450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.