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Background: Nosocomial infection or Hospital acquired infection [HAI] is an infection which is not present or incubated at the time of admission in the health care settings. It adds significant rate of morbidity and mortality in health care centers. Health care workers hold an important role in the transmission of HAI. Hands and mobile phones may be a frequent cause of transmission for nosocomial infection in a hospital. Aim: To access the spread of HAI following practice of hand hygiene and usage of mobile phones by health care worker in a tertiary care hospital. Materials and Methods: 280 samples were collected from 140 health care workers, 2 samples each i.e. one from the hand and other from the mobile phone through sterile cotton swabs, which were inoculated in Nutrient, Mac Conkey and Blood agar. Identification and speciation were done through biochemical tests. Antibiotic sensitivity testing was done using Kirby Bauer disc diffusion method. Result: Among 280 samples, 220 showed positive bacterial growth, in which the pathogenic organisms accounted to 75. Escherichia coli were highest [28.0%] among the isolates, whereas Enterococcus was the lowest [6.6%].