Background: The CDC defines a nosocomial infection as a localized or systemic condition caused by an adverse reaction to the presence of an infectious agent(s) or its toxin(s). It is an infection that occurs between 48 to 72 hours after admission of patients in the hospital or as soon after the hospital discharge and on the admission time, patients don't have this infection. Objectives: This study aimed to characterize the prevalence of nosocomial infection in Ghaem hospital, Mashhad, Iran. Methods: This retrospective study was conducted in all wards of the Ghaem hospital, Mashhad during the 1 year period (2013); the data were collected from the wards records and HIS system and analyzed by the SPSS software (version16). Results: In the present study, of total 35979 hospitalized patients in different wards of the Ghaem hospital was reported 1.1% of nosocomial infection. In the meantime, overall, the most prevalent organism was Acinetobacter baumannii with a prevalence of 37.2% and the minimum was linked to the Bacillus species with a prevalence 0.3%. The highest and lowest prevalence of the nosocomial infection was in the ICU and CCU with 49.9% and 0.3%, respectively. In general, among all wards of the mentioned hospital, the most frequent nosocomial infection was pneumonia (47.4%) and the lowest belonged to CSF (2.3%). Conclusions: In our study, the ICU ward was accounted for the highest rate of nosocomial infection, due to the critical importance of this ward. Preventive measures and survivelance system for reduction of nosocomial infections is needed.