PURPOSE: Neonates in neonatal intensive care units(NICU) have a high risk of acquiring nosocomial infection because of their impaired host defence mechanism and invasive procedures. Nosocomial infection result in considerable morbidity and mortality among neonates. This study was carried out to survey both the epidemiology of nosocomial infection in our NICU and the annual trends of pathogens. METHODS: We retrospectively reviewed culture proven nosocomial infection which occurred in our NICU from January 1995 to December 1999. The data included clinical characteristics, site of infection, pathogens, and mortality. RESULTS: Nosocomial infection rates was 9.0 per 100 NICU admissions during the five-year period. Major sites of infection were bloodstream(32.3%), skin(18.4%), endotracheal tube(17.2%), and catheter(10.6%). The most common pathogen was S. aureus(29.9%). and the others were coagulase-negative staphylococci(CONS)(14.8%), Enterobacter(12.4%), and Candida(9.0%). During the five-year period, nosocomial infection rates increased from 9.5 to 11.6 per 100 admissions with the increase of CONS, Candida, Klebsiella, and Acinetobacter baumannii. The infection rate of S. aureus decreased. Multiple episodes of nosocomial infection occurred in 26.1% of all nosocomial infections. Overall bloodstream infection rates were 3.6 per 100 NICU admissions during five years. CONS(29.1%) and S. aureus(27.1%) were the two most common pathogens. Increasing rates of bloodstream infection by CONS, Candida, Klebsiella, and Acinetobacter baumannii were observed. Bloodstream infection related mortality was 11.9%. CONCLUSION: The predominant pathogens of nosocomial infection in NICU were S. aureus and CONS. Bloodstream infection, the most frequent nosocomial infection, should be a major focus of surveillance and prevention efforts in NICU.