Objectives National prevalence estimates of Chlamydia trachomatis (chlamydia) and Neisseria gonorrhoeae (gonorrhoea) are important for providing insights in the occurrence and control of these STIs. The aim was to obtain national prevalence estimates for chlamydia and gonorrhoea and to investigate risk factors associated with infection. Methods Between November 2016 and January 2017, we performed a national population-based cross-sectional probability sample survey among men and women aged 18–34 years in the Netherlands. Individuals were invited to complete a questionnaire about sexual health. At the end of the questionnaire, sexually active individuals could request a home-based sampling kit. Samples were tested for chlamydia and gonorrhoea using nucleic acid amplification test (NAAT). Logistic regression analyses were performed for predictors of participation and chlamydia infection. Results Of the 17 222 invited individuals, 4447 (26%) participated. Of these, 3255 were eligible for prevalence survey participation and 550 (17%) returned a sample. Participation in the prevalence survey was associated with age (20+) and risk factors for STI. We did not detect any gonorrhoea. The overall weighted prevalence of chlamydia was 2.8% (95% CI 1.5% to 5.2%); 1.1% (0.1% to 7.2%) in men and 5.6% (3.3% to 9.5%) in women. Risk factors for chlamydia infections in women aged 18–24 years were low/medium education level, not having a relationship with the person you had most recent sex with and age at first sex older than 16. Conclusions Chlamydia and gonorrhoea prevalence were low in the general Dutch population, as was the participation rate. Repeated prevalence surveys are needed to analyse trends in STI prevalences and to evaluate control policies.