Hepatitis B is the most prevalent infection and a global concern that affects a large population. Healthcare workers (HCWs) are at higher risk of developing the disease, because of their regular contact with blood and body fluids. Objective of the study was to analyze all HCWs Knowledge, Attitude, and Practice (KAP) concerning hepatitis B infection and its vaccination, as well as the impact of educational training among them. A cross-sectional KAP study was done at a tertiary care hospital using a self structured questionnaire survey regarding hepatitis B infection and its vaccination among 156 HCWs. A short educational PowerPoint presentation (PPT) was given to them, followed by the same questionnaire survey. Pre and post-intervention data were collected to evaluate the effects of training on all HCWs. A Pre-assessment revealed that HCWs had inadequate KAP regarding Hepatitis B infection and vaccination. Ward attendants demonstrated the lowest grades in knowledge and practice and were identified as being at the highest risk. The instructional PPT intervention implemented in the study was proven to be highly effective. The results indicated a significant increase in knowledge and a favorable shift in the attitude towards hepatitis B infection and its vaccination among HCWs. Improvement in knowledge and attitude among all three types of HCW were extremely statistically significant after educational PPT presentation. Hence, this study recommends that increasing awareness of HBV infection and prevention through orientation and educational training, as well as routine tests and immunizations, will be beneficial.