Objectives: We aimed to clarify the accuracy of pregnant women's knowledge and understanding regarding infectious disease screening in early pregnancy and clarify the roles that should be played by health care providers in promoting the health of pregnant women and their children. Methods: A cross-sectional questionnaire survey was conducted in 25 hospitals across Japan from May 2018 to September 2019. We compared the agreement rates regarding screening results for hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, human T-cell leukemia virus-1 (HTLV-1), and cervical cytology in the medical records and understanding of their results by pregnant women. We then investigated whether participants had knowledge regarding the risk of mother-to child transmission in these diseases and factors associated with their knowledge. Results: We enrolled 2,838 respondents in this study. The rates of agreement for HBV and cervical cancer screening related to human papillomavirus infection were "substantial," those for syphilis was "moderate," and those for HCV and HTLV-1 were "fair," according to the Kappa coefficient. The rate of knowledge regarding mother-to-child transmission of syphilis was highest (37.0%); this rate for the other items was approximately 30%. Increased knowledge was associated with higher educational level and higher annual income. Conclusions for Practice: Pregnant women in Japan had generally good levels of understanding regarding their results in early-pregnancy infectious disease screening. However, they had insufficient knowledge regarding mother-to-child transmission of these diseases. Health care providers should raise awareness in infectious disease prevention among pregnant women and the general public, providing appropriate measures and implementing effective perinatal checkups and follow-ups for infectious diseases. Significance: A previous study reported biases in knowledge about mother-to-child disease transmission among pregnant women. However, there are no reports on the accuracy of pregnant women's understanding and their backgrounds related to knowledge about infectious diseases. In this report, Japanese pregnant women had generally good understanding of their results in infectious disease screening. However, they had insufficient knowledge regarding mother-to-child transmission of infectious diseases. It is crucial for health care providers to raise awareness in infectious disease prevention and provide appropriate measures for pregnant women and the general public, such as implementing effective perinatal checkups and follow-ups.