Bloodstream infections are the most frequent nosocomial infections in neonatal intensive care units. This retrospective study surveyed the epidemiologic characteristics of nosocomial bloodstream infections which occurred in the neonatal intensive care unit from January 1, 1997 to December 31, 1999. The overall infection patient rate was 5.5% in the 3-year period, and the overall infection patient-day rate was 4.4 per 1000 patient-days. Low birth weight was a risk factor for bloodstream infections. The rate of infection for neonates with birth weight below 1000 g ranged from 36.6% to 45.8% (1997: 36.6%; 1998: 45.8% and 1999: 38.9%). The most common pathogens causing nosocomial bloodstream infection were: Staphylococcus aureus (18.5%) (with 92% oxacillin-resistant), Acinectobacter baumannii (16.3%), Klebsiella pneumoniae (11.9%), Escherichia coli (9.6%), and Pseudomonas aeruginosa (8.1%). The mortality due to nosocomial bloodstream infection was highest among gram-negative bacteria, especially with P. aeruginosa (45.5%). Therefore, surveillance of nosocomial bloodstream infection and successful strategies to decrease nosocomial bloodstream infection, such as infection control and optimal antibiotic use, are warranted.