Background: Hepatitis B, caused by the Hepatitis B virus (HBV), presents a significant global health challenge, especially in regions like Bangladesh where its prevalence is intermediate. The virus is transmitted through percutaneous or mucosal exposure to infectious blood or bodily fluids, posing a substantial risk to healthcare workers. Hepatitis B can lead to severe liver diseases if untreated. In Bangladesh, the burden of Hepatitis B is high, necessitating urgent intervention, particularly among healthcare professionals such as senior staff nurses who are on the frontline of managing and preventing the disease. Methods: This cross-sectional study assessed the knowledge levels of 210 senior staff nurses at Bangabandhu Sheikh Mujib Medical University (BSMMU) regarding Hepatitis B management and prevention. A structured questionnaire, translated into Bengali, was used for data collection through structured interviews. Convenience sampling was employed, and data were analyzed using SPSS (Version 24.0). Descriptive statistics and t-tests/ANOVA were used to explore the relationship between socio-demographic characteristics and nurses' knowledge about Hepatitis B. Ethical clearance was obtained from the Institutional Review Board (IRB) of BSMMU. Results: The majority of respondents were female (70%), aged below 30 years (59.5%), and had basic nursing education (89.5%). Most had 1-10 years of work experience (89%) and earned less than 35,000 BDT monthly (78.1%). Regarding vaccination, 64.3% had completed it, 26.7% were partially vaccinated, and 9% were unvaccinated. Occupational exposure to blood or body fluids was reported by 73.3% of nurses. Knowledge about Hepatitis B infection, transmission, and prevention was generally high, with 85.44%, 89.14%, and 85.11% of nurses demonstrating good knowledge in these areas, respectively. Marital status and vaccination status were significantly associated with higher knowledge levels (p < 0.05). Conclusion: Senior staff nurses at BSMMU exhibit substantial knowledge about Hepatitis B infection, transmission, and prevention. However, misconceptions persist regarding non-transmission routes and some preventive measures. Married nurses and those with complete vaccination status demonstrated higher knowledge levels. These findings highlight the need for targeted educational interventions to address knowledge gaps and reinforce preventive practices among nurses. This study underscores the importance of continuous education and vaccination programs to enhance the effectiveness of Hepatitis B management and prevention efforts in healthcare settings.