Objective The Hepatitis B virus (HBV) poses a substantial threat to global health. Particularly, healthcare professionals, due to their exposure to blood and blood-derived products, are at an elevated risk of HBV infection. This research aimed to delineate the sero-prevalence of HBV markers (HBsAg and Anti-HBs) and identify associated risk factors among staff in the operating rooms and laboratories of Turgut Özal Medical Center. Material and Method We collated the HBsAg and Anti-HBs serological outcomes for 180 medical personnel from the afore mentioned departments during 2017. Data, encompassing demographics and potential risk factors (e.g., duration of employment, familial HBV history, blood transfusions), were procured through structured interviews. Subsequent statistical analyses were conducted utilizing the SPSS 18 software. Results A total of 180 healthcare professionals were evaluated. Of these, 81 (45%) were male and 99 (55%) were female. The average age for males was 34.5±7.8, while for females it was 35.3±6.5. Among the participants, 71 (39.7%) were nurses, 57 (31.8%) were anesthesia technicians, 35 (19.6%) were biologists/ chemists, and 16 (8.9%) were administrative staff. Only 3 participants tested positive for HBsAg, hence the statistical relationship was evaluated between the variables of the participants and the presence of Anti- HBs positivity (immunity). A statistically significant relationship (p=0.048) was only found between Anti- HBs positivity (n=152, 85.4%) and the department in which they worked. Conclusion The rate of Anti-HBs positivity among the workers was 85.4%. There was a statistically significant relationship between HBs antibody positivity and the department in which they worked. This was attributed to a higher immunity rate among laboratory workers. Healthcare professionals at risk for HBV should be screened, and those not immune to HBV should be vaccinated.