Background: Knowledge of oral health is a fundamental prerequisite for healthy behavior, allowing individuals to take measures to protect their overall health. This cross-sectional study aimed to examine the knowledge of oral health as well as to assess the oral hygiene habits among healthcare and non-healthcare students. Methods: The study was based on a questionnaire and was conducted among 1088 students. Most of the students, 67.6% were non-healthcare students. Data were processed by Mann–Whitney or Kruskal–Wallis one-way ANOVA test. Results: The correct median score and interquartile range were 11 (9–13) for all surveyed students, 11 (9–12) for students in non-healthcare programs and 13 (11–14) for students in healthcare programs. Students did not significantly differ in the knowledge of oral health by gender (p = 0.082) but did differ by age, study program, and year of study (p ≤ 0.001). Students whose family members work in the field of dental medicine also showed better oral health knowledge (p ≤ 0.001). Conclusion: The results showed good oral health knowledge among tested university students. However, it is important to emphasize that students who showed better knowledge more often used additional aids to maintain oral hygiene; therefore, the obtained data underline the importance of students’ further education in order to better understand and maintain oral health.