Background and Aim: Occupational exposure to blood-borne pathogens, mainly human immunodeficiency virus (HIV), hepatitis B (HBV) and hepatitis C (HCV), poses a serious risk to healthcare workers (HCWs), especially in developing countries, due to the high prevalence of these pathogens and fewer safety precautions. The aim of this study was to investigate the seroprevalence of HBV, HCV, and HIV infections and to measure the vaccination practices in HCWs at three tertiary care hospitals in Delhi, India. Method: In a descriptive (cross-sectional) study, the HCWs of three tertiary hospitals were selected by simple random sampling and divided into four different groups (nurses, laboratory and operational theater technicians, doctors, and housekeeping workers). The participants were screened for the presence of hepatitis B surface antigen (HBs Ag), antibody to hepatitis B surface antigen (anti HBs), antibody to hepatitis C (anti HCV), and antibody to HIV (anti HIV). From June 2010 to April 2012, a structured questionnaire was administered to 850 HCWs after obtaining consent. Results: Among 850 HCWs, 51.8% were nurses and 50.6% were female with a mean (SD) age of 34(8.7) years. The overall seroprevalence of HBsAg, anti-HCV, and anti-HIV was 1.1%, 0.3%, and 0.1%, respectively. There was a high proportion of HBsAg positivity among housekeeping workers (4.9%) followed by nurses (3.3%). Out of 9 positive cases of HBsAg, 66% (6) were never vaccinated and out of a total of 11 positive subjects, 72 % (8) had previous exposure in the workplace. Complete HBV vaccination was done in 78.2% (605) of the HCWs and 11.3% (75) were partially vaccinated. Only 20.1% had checked their anti-HBs. Protective (>10 IU/mL) anti-HBs was seen in 70.6% (600) of the participants, indicating that nearly one third of HCWs were not protected against HBV infection. The majority of the study subjects (63.6%) believed that they were immunized against hepatitis B and did not need to check the immunity titer. Conclusion: Not all HCWs were vaccinated and the majority of vaccinated subjects did not know their immunity level. Housekeeping workers had a high seropositivity rate of infections and a low rate of vaccination against HBV. Institutional policy and training were found to be of paramount importance to improve the quality of health in HCWs.