Abstract Background Prevalence data on viral hepatitis B (HBV), hepatitis C (HCV), and HIV infection in prison are often scarce or outdated. There is currently no systematic screening for these blood-borne viral infections (BBV) in Belgian prisons. There is an urgency to assess the prevalence of these BBV to inform policymakers and public healthcare. Methods This was a multicentre, interventional study to assess the prevalence of BBV using opt-in screening in prisons across Belgium, April 2019 – March 2020. Prisoners were tested using a finger prick and BBV risk factors were assessed using a questionnaire. A generalized linear mixed model was used to investigate the association between the various risk factors and HCV. Results In total, 886 prisoners from 11 Belgian prisons were screened. Study uptake ranged from 16.9 to 35.4% in long-term facilities. The prevalence of HCV antibodies (Ab), hepatitis B surface antigen (Ag) and HIV Ab/Ag was 5.0% (44/886), 0.8% (7/886), and 0.2% (2/886). The adjusted odds for ...