The objective of this study was to evaluate the knowledge and awareness of healthcare workers about the prevention and transmission of hepatitis B virus. The study was conducted in Nishtar Medical Hospital, Multan, from January 2023 to September 2023 and included 300 healthcare workers, such as doctors and nurses. The participants were asked to fill out a 50-question questionnaire designed to test their knowledge about hepatitis B. The questionnaire was in English, but assistance was provided if needed. The questions were related to the virus, behaviors, and medical practices when treating infected patients, and it took an average of 30 minutes to complete. The results showed that 74% of the healthcare workers knew the consequences and possible complications of acquiring HBV infection. Between 69% and 80% of the respondents answered correctly about the transmission routes. However, only 24% of the HCWs knew the age distribution of the infections and that neonates were at the highest risk of developing it through mother-to-child transmission. Respondents answered wrongly about transmission through food and cutlery with the infected (69%), through sneezing and coughing (81%), prevention of infection by thorough cleaning (50%), and not sharing food and cutlery with the infected (55%). Only half of the workers knew about disposing of needles and syringes correctly. The average knowledge score was 24 ± 5 with a median score of 24 (10-33). Respondents performed the worst when answering questions about CHB monitoring and treatment. In conclusion, this study found that the awareness and knowledge about hepatitis B prevention and transmission among healthcare workers in Pakistan is poor. Therefore, it is recommended to make training programs mandatory for professionals to improve disease management.