In recent years, there have been increasing knowledge gaps and biases in public health information. This has become especially evident during the COVID-19 pandemic and has contributed to the spread of misinformation. With constant exposure to disinformation and misinformation through television, the internet, and social media, even university students studying healthcare-related subjects lack accurate public health knowledge. This study aimed to assess university students' knowledge levels of basic public health topics before they started their specialized education. Participants in this cross-sectional study were first-year students from medical schools, health-related colleges, and liberal arts colleges. A self-administered electronic survey was conducted from April to May 2021 at a private university in Japan, comprising six colleges with seven programs. Data analysis, conducted from June to December 2022, included students' self-reported public health knowledge, sources of information, and self-assessment of knowledge levels. Among the 1,562 students who received the questionnaire, 549 (192 male [35%], 353 female [64.3%], and 4 undisclosed [0.7%]) responded to one question (participants' response rate for each question; 59.6%-100%). The results showed that students had limited public health knowledge, especially in sexual health topics, and 10% of students reported not learning in class before university admission the following 11 topics: two on Alcohol, Tobacco, and Other Drugs; eight on Growth, Development, and Sexual Health; and one on Personal and Community Health. These results indicate significant knowledge gaps and biases, as well as gender gaps, in public health education, especially in the area of sexual health, which may help educators and educational institutions to better understand and prepare for further specialized education. The findings also suggest a need to supplement and reinforce the foundation of public health knowledge for healthcare majors at the time of university admission.