Background: Neonatal care protocols are standardized guidelines that outline the best nursing practices for managing and treating ventilated neonates. These protocols cover various aspects of care, including respiratory management, nutritional support, medication administration, and developmental care. Aim: This study aims to assess nurses' compliance regarding the application of neonatal care protocol on mechanical ventilation in intensive care unit. Methods: A descriptive design was utilized to conduct this study. Setting: This study was conducted in the Neonatal Intensive Care Unit at El Sheikh Zayed Al Nahyan Hospital affiliated to Specialized Amana Medical Centers in Egypt. Sample: A purposive sample of 68 nurses. Tools: Nurses' Knowledge Questionnaire Sheet to assess nurses' knowledge and Observational Checklists to assess nurses' practices regarding the application of neonatal care protocol on mechanical ventilation. Results: It revealed that 54% of the studied nurses had an average level of knowledge, and 68% of the studied nurses demonstrated an incompetent level of practices regarding the application of neonatal care protocol on mechanical ventilation. Conclusion: More than half of the studied nurses had an average level of knowledge, and more than two-thirds of the studied nurses demonstrated an incompetent level of practices regarding the application of neonatal care protocol on mechanical ventilation. Moreover, there was a positive correlation between total level of nurses' knowledge and total level of nurses' practices. Recommendations: Developing a nursing protocols for mechanical ventilation, and conducting periodical educational training programs for nurses to update and enhance their knowledge and practices in caring for mechanically ventilated neonates.