Background: Ventilator-associated pneumonia is a prevalent and dangerous issue among newborns who are mechanically ventilated. It is interrelated with longer hospital stays, high morbidity and mortality rate. The study aimed to assess nurses' knowledge and practice regarding prevention of ventilator-associated pneumonia in neonates. Subjects and methods: Design: A descriptive cross-sectional design was applied in this study. Setting: The research was done the study in neonatal intensive care units at three hospitals affiliated to the universal health insurance in Port Said Governorate. Subjects: The study sample was sixty nurses working in neonatal intensive care units and forty neonates on mechanical ventilation. Tools: Data were obtained through questionnaire sheet and observational checklists. Results: The present study showed that approximately one third of the studied nurses had satisfactory total knowledge regarding ventilator associated pneumonia and its prevention while nearly two thirds of them had unsatisfactory total knowledge. Also, few studied nurses had adequate total practice score regarding prevention of ventilator associated pneumonia, while most of them had inadequate total practice score regarding prevention of ventilator associated pneumonia. Conclusion: There was a statically significant positive correlation between total score of nurses' knowledge and their total practice score about preventing ventilator associated pneumonia. Recommendation: To keep nurses' skills and knowledge up to date on the prevention of neonatal ventilator-associated pneumonia, an ongoing training program is recommended.