Background: Hand hygiene is seen as one of the principal means of preventing hospital–acquired infections for both health care workers and patients. Healthcare–associated infections (HCAIs) are worldwide primarily due to poor hand hygiene practices. Hand antisepsis reduces the prevalence of health–care–associated infections. The hands of hospital settings workers are the most common vehicle for the transmission of micro–organisms from patient–to–patient and within the healthcare environment. Healthcare–associated infections are drawing increasing attention from patients, insurers, governments, and regulatory bodies. This is not only because of the magnitude of the problem in terms of the associated morbidity, mortality and low rate of treatment but also due to the growing recognition that most of these are preventable. Aim of the study: The aim of our study was to analyze the hand hygiene among HCWs both right and left hand. Methods: The study was carried out in the Medical and Surgical ICUs of a tertiary care NIMS multispecialty and super specialty hospital, Jaipur, Rajasthan. Considering the resources available, we decided to randomly select 129 samples from each hand of 43 healthcare workers (both male and female). All the samples were collected during early hours (i.e., before entering ICUs). Processing was carried out by taking the swab samples with peptone water from each HCW and incubated for 18–24 hours at 37℃ and sub cultured on blood and MacConkey agar and again incubated the culture plates for 18–24 hours at 37℃, Gram staining and biochemical identifications were done. Result: The percentage of various isolates in right hand were GPB (67.44%), E. coli (1.55%), Pseudomonas spp. (0.78%), Klebsiella spp. (0.78%) and No growth (29.45%) and in left hand GPB (65.12%), E. coli (3.10%), Pseudomonas spp. (1.55%), Klebsiella spp. (1.55%) and No growth (28.68%). Conclusion: In conclusion, hand hygiene among healthcare workers and patients is a key issue with in a healthcare setting to prevent healthcare–associated infections.