OBJECTIVE Healthcare-associated infections in neonatal intensive care units (NICUs) are associated with a significant risk of morbidity and mortality. Knowledge regarding pathogens, primary sources of infection and antibiotic resistance in the NICU is essential for developing management strategies. This study aimed to analyze the long-term characteristics of healthcare-associated infections in a tertiary referral center in southern Taiwan. METHODS Infants < 30 days old, with positive blood, cerebrospinal fluid, urine or tissue fluid cultures during hospitalization in the NICU of National Cheng Kung University Hospital from July 1989 to June 2008 were included in the study. RESULTS In total, 1,417 organisms and episodes were identified during the study period. Gram-positive organisms, Gram-negative organisms and fungi constituted 923 (65.1%), 358 (25.3%) and 136 (9.6%) of the pathogens, respectively. Of the Gram-positive organisms, coagulase-negative staphylococci (51.5%), Staphylococcus aureus (34.8%) and Enterococcus spp. (6.1%) were the major pathogens; and 27% of Staphylococcus aureus isolates were oxacillin-resistant. For the Gram-negative organisms, Klebsiella pneumoniae (22%), Pseudomonas aeruginosa (21.8%), Escherichia coli (16.7%) and Enterobacter cloacae (16.7%) were dominant. Also, Candida albicans accounted for 50% of fungal infections. The most common source of infection was bloodstream infection (59.0%), and 5.6% of these were catheter-related. Skin and soft tissue infections were also frequent (26.3%). CONCLUSIONS Bloodstream and skin/soft tissue infections caused by commensal species play an important role in healthcare-associated infections in the NICU. New measures should be developed in response to the changing patterns in the NICU.