OBJECTIVE Evaluate the feasibility, safety, and efficacy of adjunctive treatment with dornase alfa in preterm patients with ventilator-associated pulmonary infection (VAPI) compared to standard care. WORKING HYPOTHESIS We hypothesize that therapy with dornase alfa will be safe and well tolerated in the preterm population with no worsening of symptoms, oxygen requirement, or need for respiratory support. STUDY DESIGN Prospective, randomized, blinded, pilot study comparing adjunctive treatment with dornase alfa to sham therapy. In addition to standard care, infants were randomized to receive dornase alfa 2.5 mg nebulized via endotracheal tube (ETT) every 12 hr for 7 days or sham therapy. ETT secretion gram stain and culture and chest X-ray (CXR) findings were evaluated. Respiratory support data were downloaded from the ventilator. RESULTS Fourteen infants developed VAPI between 2012 and 2014; 11 enrolled in the study. Six received dornase alfa and five received sham therapy. Average gestational age at birth was 25 weeks and age at study entry was 31 days. There were no differences in demographics, ETT white blood cell count (WBC), CXR, or mean airway pressure (MAP) between the two groups. There was a trend towards decreased oxygen requirement (FiO2) in the treatment group that did not reach statistical significance. No side effects were observed in the treatment group. CONCLUSION Treatment with dornase alfa is safe and treated infants had some improvement in FiO2 requirement but no improvement in MAP. A larger randomized trial is needed to evaluate the efficacy of this therapy. Pediatr Pulmonol. 2017; 52:787-791. © 2017 Wiley Periodicals, Inc.