Background: university students are believed to retain the highest levels of health literacy. They are perceived as the opinion leaders within their communities; therefore, their health-related beliefs and attitudes are deemed important for public health campaigns. This study aimed to investigate the COVID-19 vaccine hesitancy drivers among university students in the Czech Republic. Methods: a cross-sectional study using a self-administered questionnaire was carried out in the weeks before the unrestricted vaccine deployment to Czech adults. The questionnaire had 21 multiple-choice items stratified in 4 categories; demographic characteristics, COVID-19-related anamnesis and influenza vaccine experience, attitudes towards COVID-19 vaccination, and the possible drivers of COVID-19 vaccine hesitancy suggested by the WHO-SAGE. Results: out of the 1351 included students, 66.8% were females, 84.5% were Czech nationals, and 40.6% enrolled in healthcare programs. The overall COVID-19 vaccine acceptance level was 73.3%, 19.3% of participants were vaccine-resistant, and only 7.4% were vaccine-hesitant. Trust in the pharmaceutical industry, trust in healthcare providers, and perceived knowledge sufficiency predicted higher odds of vaccine acceptance. In contrast, media and social media, personal beliefs, immunity misconception, previous COVID-19 infection, and suspicions about novel vaccines and the local availability predicted higher odds of vaccine hesitancy. Conclusions: The findings of this study predict a fair probability to achieve community immunity (herd immunity) among the target population group. The primary prevention strategies in the Czech Republic need to be culturally sensitive and inclusive for foreign nationals. As one-quarter of the participating students are dependent on vaccine safety data, this study findings support the call for independent studies evaluating the side effects of COVID-19 vaccines.