To identify independent risk factors to develop a central line- associated bloodstream infection (CLABSI) in critically ill neonates with major underlying diseases.A nested case-control study was conducted in a neonatal intensive care unit (NICU). Patients with a central venous catheter (CVC) were included. Cases were neonates who developed a CLABSI and controls were patients without CLABSI. Variables included: perinatal history, characteristics of the catheter, installation and catheter use, surgical interventions, and hospital stay. Odds ratio (OR) and 95% confidence intervals (CI) were calculated. X2, Fisher exact, and Mann-Whitney U tests were used when appropriate. Variables with a p value ≤0.10 in the univariate analysis were introduced in a non-conditional logistic regression model.Seventy four cases and 105 controls were analyzed. Univariate risk factors were: any surgery, abdominal surgery, length of hospitalization (≥14 d), double-lumen CVC, surgical cut-down technique, complications, CVC placement in internal jugular vein, dressing type, blood transfusions, parenteral nutrition, and number of CVC manipulations (>200). In the logistic regression analysis, independent risk factors with a p value