Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.