In this study, we have prospectively recorded healthcare-associated infections (HAIs) in NICU and found incidence density as 18 infections per 1000 patient days. Of the infections, 51.3% was bacteriemia (BSI), and 45.1% was ventilator-associated pneumonia (VAP). Gram-negative microorganisms were predominant in VAP and Staphylococcus epidermidis was the leading microorganism (53.0% of BSIs) in BSIs. Multivariate logistic regression analysis showed the importance of hood O(2) use in days (RR: 1.3) and total parenteral nutrition use in days (RR: 1.09) for BSIs. Umbilical arterial catheterization in days (RR: 1.94), ventilator use in days (RR: 1.05), chest tube (RR: 12.55), orogastric feeding (RR: 3.32) and total parenteral nutrition in days (RR: 1.05) were found to be significantly associated with VAP. In conclusion, incidence density in our unit is high and Gram-negative rods are predominant similar to developing countries. These results strongly suggest improving measures of prevention and control of HAIs in the unit.