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This study was carried out to determine the knowledge and attitude of health care workers in a Federal Teaching Hospital in South West, Nigeria towards Hepatitis B vaccination and infection. This study employed a quantitative descriptive survey design, with a stratified random sampling technique. Data was collected using structured self-administered questionnaire administered to 139 health care workers. Data was analyzed using descriptive statistics. The age distribution of respondents’ showed 41% and 48.9% of the respondents were in the age range of 20-30 years and 31-40 years, respectively. Only 30.2% of the respondents believe that it is vital to recap needles after use while 79.9% believe that Hepatitis B can be transmitted as a nosocomial infection. A total of 38.8% of the respondents believed that after vaccination for Hepatitis B, it is not necessary to have a blood test to confirm immunity against Hepatitis B while 61.9% of them affirmed that 90% of adults and children who are vaccinated achieve 100% protection against Hepatitis B virus. The majority of the respondents (87.1%) were of the opinion that Hepatitis B virus is about 100 times more infectious than HIV. Almost half (48.9%) of the participants indicated that a person who has been vaccinated or recovered from previous Hepatitis B infection can still infect other. Also, 44.6% of the respondent strongly agreed that vaccination against Hepatitis B virus should be made available to all healthcare workers for free. About half (48.2%) of the respondents indicated they have not been vaccinated against Hepatitis B virus and of these only 10.8% received the completed 3 dose series. Although the health care workers claimed knowledge of Hepatitis infection, their practice of preventive measures was not commensurate with their knowledge. It is therefore imperative to improve their knowledge to influence their practice. Resources for practice of Hepatitis B preventive measures should be made regularly available to the health care workers in various health institutions to reduce the transmission of Hepatitis B among the health care workers decrease medical as well as financial burden, hence improving the management of cirrhotic patients. These predictors, however, need further work to validate reliability.