Background: Nursing students share their study time between clinical and university environments, and due to their clinical training, are at increased risk of contracting the hepatitis B virus (HBV). This study investigated the vaccination coverage, knowledge, attitudes, and practices of nursing students regarding HBV in Greece. Methods: A cross-sectional study was conducted from September 2022 to July 2023, using an anonymous self-administered questionnaire, with informed consent obtained from each participant. Results: A total of 1261 nursing students from University Institutions of the Attica region participated in the convenience sampling. The response rate of the nursing students was 68.6%. A total of 27.7% of the sample was fully vaccinated against HBV. The reasons for not accepting the vaccine were not found to be related to participants’ level of knowledge or attitudes (p > 0.05). The knowledge score ranged from 0% to 96.9%, with a mean of 62.2% (SD = 17.0%). Multivariate linear regression showed that longer year of nursing education was associated with better practices and attitudes towards HBV (p < 0.05). Conclusions: The emergence of low vaccination coverage of nursing students against HBV identifies the need for departments of nursing studies to proceed in the development of educational and intervention programs on infection control. This study was not registered.