BackgroundDrug overdose is the major cause of morbidity and mortality among persons who inject drugs (PWID). We assessed factors associated with the non-fatal drug overdose among PWID in three Croatian cities and national trends of overdose-related mortality (OM), and rates of uptake of opioid agonist drug treatment (OAT). MethodsWe used a respondent-driven sampling method to recruit 830 PWID in Zagreb, Split and Rijeka in 2014/2015. Participants completed behavioural questionnaires that included questions about overdose history, and we used Poisson regression to assess factors associated with self-reported overdose. We used joinpoint regression to calculate national trends of OM from 2001 to 2015 and rates of uptake of drug treatment from 2005 to 2015. ResultsLifetime prevalence of self-reported drug overdose in our RDS sample was 45.2%, while 4.1% of PWID reported overdose in the past 12 months; PWID who injected more than one type of drug in the past 12 months (adjusted prevalence ratio [aPR] = 4.56, 95% confidence intervals [CI] = 1.35–15.38) compared to injecting only heroin, and those enrolled in OAT (aPR = 1.94, 95% CI = 1.01–3.74) were more likely to report overdose in the past 12 months. We observed an increase in annual percent change (APC) of the national OM rates from 2001 to 2007 (APC = 22.5%, 95% CI = 16.3–29.0) and a decline from 2007 to 2015 (APC = −8.0%, 95% CI = −5.3- −10.5). The national rates of drug treatment enrollment increased from 2005 to 2010 (APC = 12.0%, 95% CI = 10.3–13.8), mostly due to increase in provision of buprenorphine from 2005 to 2008 (APC = 130.4%, 95% CI = 102.1–162.7). ConclusionInjecting more than one type of drugs and enrollment in OAT while still injecting drugs was positively associated with non-fatal overdose in our sample. To further reduce OM in Croatia we suggest improvements in coverage and delivery of OAT and establishment of provision of naloxone for PWID.