Objectives To investigate the impact of harm-reduction programmes on HIV and hepatitis C virus (HCV) incidence among ever-injecting drug users (DU) from the Amsterdam Cohort Studies (ACS). Methods The association between use of harm reduction and seroconversion for human immunodeficiency virus (HIV) and/or hepatitis C virus (HCV) was evaluated using Poisson regression. A total of 714 DU were at risk for HIV and/or HCV during follow-up. Harm reduction was measured by combining its two most important components—methadone dose and needle exchange programme (NEP) use—and looking at five categories of participation, ranging from no participation (no methadone in the past 6 months, injecting drug use in the past 6 months and no use of NEP) to full participation (≥ 60 mg methadone/day and no current injecting or ≥ 60 mg methadone/day and current injecting but all needles exchanged). Results Methadone dose or NEP use alone were not associated significantly with HIV or HCV seroconversion. However, with combination of these variables and after correction for possibly confounding variables, we found that full participation in a harm reduction programme (HRP) was associated with a lower risk of HIV and HCV infection in ever-injecting drug users (DU), compared to no participation [incidence rate ratio 0.43 (95% CI 0.21–0.87) and 0.36 (95% CI 0.13–1.03), respectively]. Conclusions In conclusion, we found that full participation in HRP was associated with a lower incidence of HCV and HIV infection in ever-injecting DU, indicating that combined prevention measures—but not the use of NEP or methadone alone—might contribute to the reduction of the spread of these infections.