BACKGROUND: Nosocomial infection is an infection acquired in hospital by a patient who was admitted for a reason other than that infection. Nosocomial acquisition of infection is now the most common mode of transmission of infection in neonatal intensive care units. There is limited information on the prevalence of nosocomial infection in Ethiopia particularly in the pediatrics population specifically in the neonates. OBJECTIVE: The objective of this study was to determine the prevalence and identifying associated factors of neonatal nosocomial infection in those admitted at Tibebe-Ghion Specialized Hospital NICU; from Feb1/2019 to June 30/2020. METHOD: An institution based cross sectional study was conducted in TibebeGhion specialized hospital in the neonatal care unit from Feb1/2019 to June30/2020 in all neonates admitted to the hospital neonatal unit and stayed more than 48hours were included in the study. During the study period 752 neonates who fulfilled inclusion criteria were included; from which 384 were taken by random sampling. Data were collected on nosocomial infection, socio-demographic characteristics, underlying diagnoses, and therapeutic interventions/treatments by well-prepared abstraction sheet by principal investigator and trained residents and nurses. The data was cleaned and checked for completeness, and entered into SPSS version 23 soft ware package for analysis. RESULTS: Out of 384 neonates; 104 neonates had blood culture, 92 had blood culture identified nosocomial infection and the prevalence of nosocomial infection is 27.0 %( 95%CI: 20.1-28.6).Fifty eight (63%) of the isolated organisms were gram negative. The most common organisms identified were klebsilla pneumonia 36 (39.11%) followed by coagulase negative staphylococcus 21 (22.83%).Length of hospital stay before diagnosis, intervention with nasogastric tube and presence of congenital anomalies were significantly associated diagnosis of nosocomial infection. But gestational age and birth weight were not statistically associated with nosocomial infection. CONCLUSION: This study shows that there is a high prevalence of nosocomial infection and was associated with prolonged hospitalization, invasive intervention like nasogastric tube insertion. Decreasing duration hospitalization and invasive procedure may decrease the occurrence of neonatal nosocomial. I would recommend on improving standard of care by training staffs or by adhering a protocol that can decrease that nosocomial infection. A more focused study is required on factors leading to nosocomial infection in neonates’ intensive care unit setting. Key words: Neonates; Nosocomial Infection; Risk Factors; Neonatal Intensive Care Units