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Nosocomial infection is one of the leading causes of death and increased morbidity for hospitalize patients. It occurs after the exposure to health care and might be exogenous; due to surgical invasive procedure, organ transplant and implanted foreign bodies from health care professionals and environment or endogenous; immune compromised individual, age (elderly and neonate) this infection causes prolong hospital stay and account for increased risk factor of morbidity, mortality treatment failure and high cost of care giver. The aim of the study was to determine the knowledge and utilization of measures for the prevention of nosocomial infection among nurses in a tertiary institution Benin City, Edo State, Nigeria. . The study was carried out using a descriptive cross sectional study a non-experimental design. A total of two hundred and fifty seven nurses were sampled from the population of nurses. A multi-staging sampling technique was used to stratify the wards into units and wards were selected randomly by balloting. Data was gotten from a well-structured questionnaire and a proforma was used to assess the knowledge and utilization of measures for preventing nosocomial infection in selected wards. The result from the study showed that majority (74.3%) of the respondents exhibited correct knowledge of utilization of measures for preventing nosocomial infections. Findings on levels of utilization of measures for preventing nosocomial infection revealed that the respondents have high utilization of measures for preventing nosocomial infections. Also Study findings in respect to factors influencing the measures utilized for preventing nosocomial infection revealed that unavailability of personal protective equipment, hospital policies, emergency situations, years of practice, attitude of nurses and prompt supervision by infection control unit are all factors influencing utilization of measures of preventing nosocomial infection. The hypotheses tested showed that there is a significant relationship between the utilization of measures for preventing nosocomial infections among nurses and their years of practice. Hence, Interventions can then be designed to improve the quality of nursing services by offering basic information such as the ones provided in this study.