Neonatal sepsis is a major cause of death in newborns despite sophisticated neonatal intensive care. This retrospective study reviewed the clinical characteristics of cases of culture-proven sepsis in a neonatal intensive care unit from January 1992 to December 2001. Patients were divided into those with onset of sepsis in the first 7 days of life (early-onset group) and those with onset after the seventh day of life (late-onset group). A total of 270 cases with 325 episodes of sepsis and 353 isolated pathogens were identified and included in the study. The male-to-female ratio was 1.4. The majority of cases of sepsis occurred in low birth weight (75.9%) and premature babies (76.7%). Late onset occurred in 71.9% of cases. Patients with late onset had a lower mortality rate than those with early onset (11.3% vs 28.9%). Coagulase-negative staphylococci (20.1%) was the most common organism isolated, but infection with Pseudomonas aeruginosa was associated with the highest morality rate (55.0%). Late-onset sepsis was significantly more common in very low birth weight and premature infants. The most frequently encountered pathogens in the early-onset group were group B streptococci (GBS) and Escherichia coli, while in the late-onset group, the organisms were coagulase-negative staphylococci and Enterobacteriaceae, including E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii. GBS infection resulted in the highest mortality when the onset of sepsis was within the first 24 hours of life.