Similar to some other Central European countries, Croatia has low HIV prevalence among injecting drug users (IDUs) but high hepatitis C (HCV) prevalence. This may indicate different patterns of risk behaviour in this region than in other parts of Europe. The main objectives of this study were to assess the seroprevalence of HIV and hepatitis B and C and related risk factors among IDUs in the three largest Croatian cities (Zagreb, Split, Rijeka) and within the national prison system, as well as to apply a multiplier-method population size estimation of IDUs in Zagreb, Split and Rijeka. Recruitment sites were selected in collaboration with the local public health institutes, NGOs, Centers for treatment municipalities and the judiciary system. Participants were recruited during September and October 2007. Trained peer-recruiters were used to recruit IDU participants at treatment and harm reduction centres as well as pre-identified social, commercial and street based venues. Participants completed the study questionnaire and provided venous blood samples for HIV, hepatitis B and hepatitis C testing. The study included 601 participants, of whom 121 were recruited in Split, 130 in Zagreb, 150 in Rijeka and 200 in the prison system. The prevalence of positive anti-HCV tests was 65% in Split, 51% in Zagreb, 29% in Rijeka and 44% in the prisons. The prevalence of anti-HBcAg was 31% in Split, 13% in Zagreb, 9% in Rijeka and 24% in prison. No case of HIV infection was found. The estimated IDUs population sizes were 2,805 for Zagreb area, 3,347 for Split and 1,370 for Rijeka area, however confidence intervals were very large, indicating the need for larger samples. A high frequency of positive markers on hepatitis B virus and C virus in the population of injecting drug users in Croatia has been confirmed with this research, as well as a low prevalence of HIV infection. This may be related to relatively low levels of injecting risk behaviour and injecting frequency although it is not possible to make strong conclusions on risk behaviour, as participants were mostly recruited in harm reduction programmes. This research should be followed by targeted activities for reducing risks of infectious diseases among injecting drug users in the Republic of Croatia and future research at the national level.