Background: Globally, health workers are at risk of contracting Hepatitis B infection if preventive measures are jeopardized. This study assessed the knowledge and practice of prevention of Hepatitis B, identified factors affecting practice of HBV prevention among health care workers (HCW) and assessed factors influencing vaccination against hepatitis B in Wesley Guild Hospital, Ilesa, Osun State, Nigeria. Method: A descriptive cross sectional design was adopted. One hundred and ninety-two health care workers were selected using simple random sampling technique. Data were collected using a structured questionnaire and was analyzed using SPSS version 20.0 Result: Findings showed that 99.5% of the respondents had good knowledge about HBV infection, 99% of the respondents had good knowledge on prevention of HBV infection. Knowledge was associated with academic qualification (P = 0.001). Also, 60.9% had good practice of prevention of hepatitis. Years of experience was not associated with practice of prevention of HBV Infection (P = 0.056). Also, 85.4% of health workers had been tested for hepatitis virus before and less than three – quarter had received HBV vaccine. Also, (29.2%) had never received hepatitis B vaccine. Barriers associated with HBV vaccination among HCW include: availability of hepatitis B vaccine in the hospital (87.5%), cost of the vaccine (76%) fear of needle prick during vaccination (50%). Conclusion: The study concluded that majority of the health care workers had good knowledge of Hepatitis B prevention but not all of them with good knowledge carried out good practices regarding to HBV infection prevention.
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