Background: The pathogenic bacterium on the hands of healthcare workers (HCWs) is considered as the main route of spread of nosocomial infections. The study was aimed to determine the bacterial contamination of hands of HCWs during routine patient care in a large teaching hospital. Material and Methods: This study includes a total of 200 consecutive, non-repetitive participants. Doctors, medical students, nurses and attendants from the Department of Anaesthesia and Department of Medicine of the hospital were included, who were actively providing health care. The informed consent was taken from all the participants of the study. The hand impressions of the participants were taken on 5% blood agar and processed as per guidelines. After taking the hand impressions, hand cleaning of each participant was done by standard preparation of 70% isopropyl alcohol-based hand rub. The hand impressions from the participants were again taken on 5% blood agar plates. Results: Out of 200 samples collected from HCWs, 95 (47.5%) samples showed growth of microorganisms. Most commonly isolated microorganism was Staphylococcus aureus present in 70% of nurses, 60% of students and 40% of attendants. Prevalence of Acinetobacter spp. was also found to be high among nurses (20%), followed by attendants, sweepers (16%) and doctors (4%). Conclusions: Hands of hospital staff are colonised with pathogenic bacteria, especially Gram-positive such as S. aureus. Hand hygiene is an effective method of reducing the bacterial flora on the hands of the HCWs.