BACKGROUND: Chlamydia trachomatis infection in pregnancy may lead to adverse pregnancy outcomes. In the Netherlands, testing for C. trachomatis is based on risk assessment. We assessed midwives' knowledge, test practices, assessment of risk behavior, and attitudes regarding testing for C. trachomatis infection during pregnancy. We evaluated the association between midwives' characteristics and their knowledge of C. trachomatis infection in terms of symptomatology and outcomes. METHODS: This was a cross-sectional study among primary care midwives in the Netherlands. Between September and November 2011, midwives from all Dutch primary care midwifery practices were invited to complete a questionnaire about C. trachomatis infection. RESULTS: Of the 518 midwives invited to participate in this study, 331 (63.9%) responded. The overall median knowledge score for questions about symptomatology and outcomes was 10 out of a maximum score of 15. The median knowledge score was higher among midwives in urban areas. In total, 239 (72.2%) midwives reported testing pregnant women for C. trachomatis. The primary reason for testing was a request by the woman herself (96.2%), followed by symptoms of infection (89.1%), risk behavior (59.3%), and risk factors for infection (7.3%). Almost 25% of midwives showed positive attitudes towards universal screening for C. trachomatis. CONCLUSIONS: Midwives were knowledgeable about symptoms of infection, but less about outcomes. Midwives test pregnant women for C. trachomatis mainly on the women's request. Otherwise, testing is based on symptoms of infection rather than on known risk factors. This may contribute to under-diagnosis and under-treatment, leading to maternal, perinatal, and neonatal morbidity.