BACKGROUND: Toxoplasmosis, listeriosis and cytomegalovirus (CMV) can negatively affect pregnancy outcomes, but can be prevented by simple precautions of pregnant women. Literature suggests that pregnant women are not always adequately informed by their care provider about preventable infectious diseases and most pregnant women have a low level of knowledge regarding these topics. There is not much information about the actual risk behaviour of pregnant women. The purpose of this study was to assess knowledge and risk behaviour related to toxoplasmosis, listeriosis and CMV infection prevention in pregnant women. METHODS: A cross-sectional survey among pregnant women from twenty midwifery practices across the Netherlands that participated in the DELIVER study, between October 2010 and December 2010. The questionnaire items covered respondents' knowledge of preventive practices in general, risk behaviour, and sources of received information. RESULTS: Of the 1,097 respondents (response 66.0%), 75.3% had heard, read or seen information about toxoplasmosis, 61.7% about listeriosis and 12.5% about CMV. The majority reported having heard about these infections from their care providers or read about these in printed media or on the Internet. Respondents showed limited knowledge about preventive practices for toxoplasmosis, listeriosis or CMV infection. Regarding toxoplasmosis, risk behaviour was more prevalent among respondents who had a high level of education, had the Dutch nationality, did not take folic acid during their first trimester, and had ever worked in a children day-care setting. Regarding listeriosis, risk behaviour was more prevalent among respondents who where in their third trimester. Regarding CMV infections, risk behaviour was less prevalent among respondents who were in their third trimester of pregnancy. CONCLUSION: Of the respondents, a substantial part did not have knowledge about preventive practices to avoid listeriosis, toxoplasmosis and CMV infections during pregnancy. Many pregnant women are appropriately avoiding risk behaviour, without knowing what they are avoiding. Advising pregnant women about behaviours and life-style habits to prevent infectious diseases remains important and information about preventive practices need to be complete and adequate. However, it may be less important to give pregnant women specific infectious diseases information. More attention towards CMV is necessary.