Background Antenatal screening for HIV, syphilis and HBV has been successfully implemented in The Netherlands, but data on other STI among pregnant women or male partners are limited. Our objectives: (i) to assess the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) among pregnant women and male partners, (ii) to identify risk factors for these STI during pregnancy, and (iii) to identify adverse perinatal outcomes (APO) associated with STI. Methods Cross-sectional study. Pregnant women aged ≤ 30 years (n = 548) and male partners (n = 425) were included at 30 midwifery practices during 2012–2016. Participants provided a self-collected vaginal swab (women) or urine sample (men) and completed a questionnaire. Perinatal data were derived from pregnancy cards. APO was defined as premature rupture of membranes, preterm delivery, low birthweight, stillbirth, neonatal conjunctival and respiratory infections. Data were analysed by logistic regression. Results STI were present in 2.4% of pregnant women (CT 1.8%, NG 0.4%, TV 0.4%), and in 2.2% of male partners (CT 2.2%, NG 0.2%, TV 0%). Of young women (≤ 20 years), 12.5% had a CT infection. Prevalent STI during pregnancy was associated with female young age (≤ 20 years vs ≥ 21 years) (adjusted OR 6.52, CI 95%: 1.11–38.33), male non-Western vs Western background (aOR 9.34, CI 2.34–37.21), and female with ≥ 2 sex partners < 12 months vs 0–1 (aOR 9.88, CI 2.08–46.91). APO was not associated with STI, but was associated with female low education (aOR 3.36, CI 1.12–10.09), complications with previous newborn (aOR 10.49, CI 3.21–34.25 vs no complications) and short duration (0–4 years) of relationship (aOR 2.75, CI 1.41–5.39 vs ≥ 5 years). Small-for-gestational-age was not associated with STI, but was associated with female low education (aOR 7.81, 2.01–30.27), female non-Western background (aOR 4.41, 1.74–11.17), and both parents smoking during pregnancy (aOR 2.94, 1.01–8.84 vs both non-smoking). Conclusions Prevalence of STI was low among pregnant women and male partners in midwifery practices, except for CT among young women. The study could not confirm previously observed associations between STI and APO, which is probably due to low prevalence of STI, small study sample, and presumed treatment for STI.