Background and aims Major declines in HIV and hepatitis C and B virus (HCV/HBV) incidence among people who inject drugs (PWID) have been attributed to early implementation of harm‐reduction programs (HRP) in the Netherlands but alternative factors such as selective mortality and demographic and drug markets shifts over time likely contributed to observed incidence declines. We quantified and tested the effect of HRP participation on risk of these infections among PWID in Amsterdam, the Netherlands. Design We emulated the design of a hypothetical, ideal randomized trial using observational data from the Amsterdam Cohort Studies (1985‐2014). Setting Amsterdam, the Netherlands Participants We included PWID who ever used opioids, had a recent history of injecting drug use (IDU) and tested negative for HIV, HCV, or HBV. Of 983 participants, 640, 137 and 308 were included for the HIV, HCV and HBV analyses and 59, 45 and 49 seroconversions were observed, respectively. Interventions Intervention arms were: complete HRP participation (≥60 mg/day methadone and 100% needle and syringe program (NSP) coverage, or any methadone dose if no recent injection drug use) versus no HRP and partial HRP participation combined (<60 methadone mg/day and/or <100% NSP coverage). Measurements Separately for each infection, we estimated the hazard ratios (HR) comparing HRP arms using marginal structural models. Findings Compared with no/partial HRP participation, complete HRP participation led to lower risk of HIV (HR=0.54, 95%CI:0.27‐1.08), HCV (HR=0.16, 95%CI:0.06‐0.40) and HBV (HR=0.28, 95%CI:0.13‐0.61) acquisition. Conclusions Complete participation in harm reduction programs appears to have led to substantial decreases in HIV and hepatitis C and B virus acquisition risk among people who inject drugs in the Netherlands. Funding AidsFonds (Project number 29703), National Institute for Public Health and the Environment (the Netherlands), and Boston University/Brown CFAR P30 AI042853 (USA).