Objective: In this study, we prospectively investigated all patients hospitalized in the neonatal intensive care unit (NICU) and assessed the etiological factors and clinical, laboratory and treatment data of the patients who experienced nosocomial infections (NI). Material and Methods: The study was conducted prospectively between January 1, 2005 and January 1, 2006 in NICU of Eskisehir Osmangazi University Faculty of Medicine, Department of Pediatrics. Results: Throughout the study period, 545 patients were assessed. A total of 133 episodes of NI developed in 120 patients. Nasocomial infection rate was 24.4% and incidence density was 21.3/1000 patient days. Univariate analysis revealed low birth weight, low gestational age, presence of respiratory distress styndrome and perinatal asphyxia were associated with high risk of NI development. Multivariate logistic regression analysis showed that patients who developed NI had significantly higher rate of antibiotic use (p=0.002), surgical interventions (p=0.015), orogastric tube placement (p=0.023) and longer period of hospitalization than those who did not. Causative microorganism was identified in 78/133 (58.6%) of the episodes. Most frequently identified microorganisms were S.aureus (21.8%), E. call (17.9%), Enterobacter species (11.5%) and coagulase negative staphylococci (10.2%). Conclusion: Multivariate analysis in our study showed that antibiotic use, the presence of surgical intervention, orogastric tube placement, and longer stay in hospital were significant risk factors for NI development. Depending on these data, new strategies should be developed to prevent NI in NICU.