Background: Lack of Cytomegalovirus (CMV) knowledge among healthcare professionals has been proven to be the main threat to pregnant women’s awareness, preventing them from reducing the risk of infection. The aims of this study were to assess the knowledge and practices of French-speaking Swiss perinatal professionals in terms of CMV prevention, as well as the sociodemographic-professional factors that influence them. Methods: This observational study used a cross-sectional design to collect data-via an anonymous electronic questionnaire in French distributed to gynecologists-obstetricians, general practitioners and midwives via various channels: e-mails and social networks of partner centers, professional associations, and conferences. The 41-item questionnaire collected data on sociodemographic and professional characteristics, general CMV knowledge, national recommendation knowledge and prevention practices. Univariable and multivariable analyses were performed. Results: A total of 110 gynecologist-obstetricians, 5 general practitioners and 226 midwives participated in the study. While more than 80% of practitioners were familiar with protective hygiene measures, significant gaps were highlighted concerning the transmission routes, as well as the signs of short- and long-term congenital CMV infection. Regarding practice, 63.3% of participants provided information on CMV to their patients, mainly during the first antenatal visit. Among those who did not, lack of knowledge and forgetfulness were the two main reasons cited. Concerning systematic screening, 45.7% of participants offered it to their patients, and 37.3% only offered it to “at risk” groups. The existence of national guidelines on CMV was known by 62.0% of participants. Multivariable analysis revealed that working as a gynecologist-obstetrician was independently associated with higher score of preventive practices, while performing ultrasound or preconception consultations was independently associated with a higher score of general CMV knowledge, and working in a university hospital was independently associated with a higher score of Swiss recommendation knowledge. A level of training higher than the basic medical or midwifery diploma and participation in fetal medicine symposia both promote a higher score of CMV knowledge and prevention practices in line with current recommendations. Conclusion: This study confirms the significant gaps in CMV knowledge among French-speaking Swiss caregivers along with the heterogeneity of their prevention practices. To raise awareness among pregnant women and reduce the burden of congenital CMV infections, improving professional knowledge through access to specific training and standardizing practices should be a national priority.