Objectives Chemsex (i.e., drug use during sex) is practiced by some men who have sex with men (MSM) and is associated with high-risk behavior. In a cross-sectional study at the sexually transmitted infection (STI) clinic of Amsterdam, we explored chemsex practices, risk behavior, and STI prevalence. Method A survey on chemsex (γ-hydroxybutyrate, crystal methamphetamine, and/or mephedrone) was offered to clinic clients during routine STI screening and to Amsterdam users of a gay online dating app. Associations were assed using χ 2 test and multivariable regression. Results Chemsex in the past 6 months was practiced by 866 (17.6%) of 4925 MSM clients and by 159 (1.5%) of 10857 non-MSM clients. Among gay dating app users, the proportion that reported chemsex engagement was higher than among MSM visiting the STI clinic (29.3% [537/1832] vs. 17.6%; P < 0.001). Chemsex was a significant risk factor for bacterial STI in HIV-negative MSM visiting the STI clinic (adjusted odd ratio, 1.5; 95% confidence interval, 1.2–1.8), but not in HIV-positive MSM. A majority practiced chemsex once a month or less, and 87.0% reported sex without drug use in the past month. Conclusions In Amsterdam, chemsex is frequently practiced and significantly associated with bacterial STI in HIV-negative MSM but not in HIV-positive MSM. Future prevention strategies to reduce STI incidence should especially target HIV-negative MSM engaging in chemsex.