Background: Ventilator-associated pneumonia (VAP) is the second most common nosocomial infection in neonates, admitted to neonatal intensive care units (NICUs). The aim of this study was to identify the main causes of bacterial colonization in endotracheal tubes and tracheal secretions of neonates hospitalized in the NICUs of our university hospitals. Materials and Methods: This cross sectional study was conducted during 2015- 2016. Thirty-nine neonates, who were under mechanical ventilation in the NICUs of Baqiyatallah and Najmiyeh hospitals of Tehran, Iran, were assessed. The patients were selected using the census sampling method. Gestational age, birth weight, duration of mechanical ventilation, length of hospital stay, tracheal discharge culture, endotracheal tube culture, blood culture, and chest radiography were evaluated. Results: In a total of 39 neonates (50.3% males and 49.7% females) with the mean age of 1.17±1.12 days, bacterial growth was reported in 6 (15.3%) cases. The antibiotic study of tracheal secretion and endotracheal tube cultures showed that 2.6% of patients were resistant to cephalosporins, aminoglycosides, nitrofurantoin, and carbapenem. Moreover, 12.8% were also resistant to fluoroquinolones, besides these antibiotics. Conclusion: Tracheal secretion culture is a simple and proper approach for the diagnosis of VAP. Besides reducing the costs, this method can play a significant role in decreasing the incidence of infections.