Objective: Neonates admitted to the Neonatal Intensive Care Unit (NICU) are at high-risk of developing nosocomial infection. Ongoing infectious disease surveillance is essential to minimise the occurrence of nosocomial infection. To determine the incidence of nosocomial infection, distribution of pathogens, associated risk factors and outcomes, we evaluated a cohort of 263 neonates who were admitted to NICU from May 2005 to May 2007. Methods: The Singapore General Hospital neonatal database was tapped into to obtain information on all nosocomial infections that occurred during the review period. The inpatient case notes of these infants were reviewed, detailed data on epidemiology of infection, results of blood and cerebral spinal fluid culture were collected; associated risk factors and outcomes were evaluated. Results: Out of 255 neonates who were analysed, 20 infants (7.8%) developed nosocomial infection. The vast majority of infections (85%) were caused by Gram-positive organisms with coaugulase-negative Staphylococci accounting for 55% infection. Birth weight and gestational age were inversely related to rate of infection (13% of very low birth weight infants [VLBW] versus 4.1% of non-VLBW infants). Among VLBW infants, prolonged mechanical ventilation, central line insertion and chest tube insertion were associated with an increased rate of late-onset sepsis (LOS). Neonates with LOS had a significantly prolonged hospital stay (mean length of stay 84 days versus 54 days). The mortality rate for nosocomial sepsis was 5%. Conclusion: This study documents the high prevalence of nosocomial infection in neonates admitted to NICU, particularly VLBW infants. Identification of VLBW infants who are at high-risk for LOS and early detection of LOS remain the keys to successful management of this condition.