Summary Health-related quality of life (HRQOL) is impaired in chronic viral hepatitis and a direct role of the virus, although suggested, has not been demonstrated. Our aim was to evaluate HRQOL at blood donation before knowledge of the diagnosis of both hepatitis C virus (HCV) and hepatitis B virus (HBV) so as to elucidate this matter. Methods Prospectively, 67 sequential patients, 35 with HCV and 32 with HBV, and 67 matched controls were administered the generic Short Form-36 (SF-36) questionnaire. After knowledge of diagnosis, the SF-36 was repeated and a disease-specific questionnaire (Liver Disease Quality of Life, LDQOL-1.0) was also administered. The Wilcoxon test and Mann-Whitney U were used for between-group comparisons. Results Before knowledge of diagnosis, patients with HCV had worse HRQOL than controls, with statistically significant changes in 7/8 domains of the SF-36, and also in its physical and mental components. In the HBV group, only 2/8 domains and the physical component were significantly different from controls. After diagnosis, similar changes persisted in the HCV group, whereas two more domains were compromised in the HBV group. Comparisons between the HCV and HBV groups did not show significant differences. Conclusion The finding of greater HRQOL impairment in the HCV group before diagnosis confirms the theory that the presence of HCV in the early stage of the disease is associated with worse quality of life.