Introduction  Hepatitis B virus (HBV) is probably the only vaccine-preventable virus transmitted from one person to the other by blood transfusion, sex, and contact with blood and blood products. HBV is highly transmissible, where the infection has been noted to transmit among the household contacts. HBV is also transmitted from the mother to the child through the transplacental barrier. Clinical infection with HBV may be chronic and could remain for a lifetime. Most exposures with HBV are automatically resolved, but a few infected people may become carriers and may transmit infections. Although HBV can be treated, complete elimination of the virus and the morbidity and mortality associated with chronic infection should be considered as a cause of serious concern. Because healthcare workers are predisposed to HBV infection, adequate knowledge about the virus and the vaccine to prevent the infection is necessary. This study is carried out to assess the knowledge of HBV infection and the status of vaccination among medical, paramedical students, laboratory technicians, and doctors. Methods The study included 256 participants attending a tertiary care teaching hospital in Telangana, South India. The participants belonged to three groups, the MBBS students (first, second-, and third-year students), the doctors (the postgraduates, medical teachers, and the clinicians), and the paramedical personnel. All the participants in the study were included after oral consent, and the study was approved by the Institutional Ethics Committee. A questionnaire containing 13 points was used for the study. Seven questions were asked to know the respondent's knowledge of HBV infection, and the other six were used to know the participant's knowledge and status of HBV vaccination. The study participants filled in the responses with their current understanding of the HBV infection and the vaccine. All the responses were analyzed using Microsoft Office Excel and drawing means and percentages. Results Among the 94 medical students, 79 (84%) knew about HBV infection. There was a significant improvement in the knowledge of HBV infection among MBBS students, with first-year MBBS (68%) to the final-year MBBS (100%). The knowledge of HBV among the doctors (postgraduates, medical teachers, and clinicians) was 100%. Among the paramedical participants that included the laboratory technicians and the nursing students, all (100%) knew about HBV infection. Very few MBBS students (12%), 28% of paramedical persons, and 45% of doctors were tested for HBV infection. The knowledge of HBV vaccination was best among the doctors (100%) followed by the paramedical personnel (89%) and the MBBS students (72%). The teaching faculty including the postgraduate students (83%) were vaccinated followed by the paramedical persons (66%), and only 24% of MBBS students were vaccinated. Conclusions The study participants had a reasonably good knowledge of HBV infection, and low vaccination rates were observed among various participants. There is an urgent need to understand the significance of HBV infection, especially among healthcare workers. Being easily transmissible and because of the availability of an effective vaccine, healthcare workers should be adequately vaccinated to prevent the spread of infection.