BACKGROUND Proper hand hygiene is the single most important, simplest, and least expensive means of reducing the prevalence of health care association infection (HAIs) and nosocomial infection. Several studies have demonstrated that hand washing virtually eradicates the carriage of infections in intensive care units (ICUs). For more than 150 years, hand hygiene has been researched in the hospital environment as a way of preventing transmission of bacteria, infection and death. The purpose of this study was to import the significance of the hand hygiene and to improve the skills in performing hand hygiene among mothers of neonates admitted in neonatal intensive care unit (NICU) at tertiary care hospital. METHODS A self structured knowledge questionnaire (KQ), practice questionnaire (PQ) and practice checklist (PC) was administered. The one to one method video on hand hygiene was played by using a laptop for about 15 minutes as an intervention with demonstration done by investigator and return demonstration was done by mothers, post test was conducted on 3rd day and 5th day followed by the intervention. RESULTS The mean scores of knowledge showed improvement in post test than the pre test in both the groups, but there was an increase in practice mean score between the groups which showed an improvement in the study group, it was higher than the control group which was statistically significant at P < 0.001. Overall, there were minimal positive correlations between the level of knowledge and practice for the study group in post-test and post-test II. Minimum positive correlation was evident but there was no significance. CONCLUSIONS This study concludes that it enhanced the knowledge on hand hygiene among mothers of neonates and it helped mothers to understand the importance of hand hygiene, when to do, how to do, why to do. Thus, it expected directly or indirectly to reduce the infection rate among neonates admitted in NICU. Further, the nurses have a great responsibility in ensuring parents adherence to hand hygiene. KEY WORDS Mothers of Neonate in NICU, Hand Hygiene, Video cum Demonstration, Mothers KPC.