Endotracheal tube (ETT) suctioning is one of the most common airway procedures applied for mechanically ventilated neonates, However, if the standard guidelines for suctioning were followed the risks of developing complications due to mechanical ventilation decreases. Aim. Evaluate the effect of clinical guidelines of endotracheal tube suctioning on nurses' knowledge and practice at neonatal intensive care unit. Research design: Quasi-experimental research design was used. Sample: A convenient sample of all the available nurses (40). Setting: The study was conducted at Neonatal Intensive Care Unit (NICU) at Minia University Hospital for Obstetric and Pediatrics (MUHOP). Tools: Two tools were used. Tool Ӏ: Included the following parts. Part Ӏ: Personal and professional characteristics of nurses. Part ӀӀ: knowledge about endotracheal tube suctioning. Tool ӀӀ: Observational checklist for endotracheal suctioning procedure. Results: The current study results proved that; the total mean score of nurses' knowledge regarding endotracheal tube suctioning was improved from 12.1 in pretest to 19.1 in the immediate posttest and 18.2 in the follow up test after three months respectively. And also, the total mean score of practice show improvement in immediate posttest and the follow-up test after three months compared to the pretest. Also, there was a positive association between nurses' knowledge about endotracheal tube suctioning with their age and years of experience. Conclusion: Providing the nurses with endotracheal suction guidelines improved their knowledge and practice mean scores regarding endotracheal suctioning in immediate posttest and follow-up test compared to the pretest. Recommendations: Hospital administration should adopt the clinical guidelines of endotracheal tube suctioning in NICU to be practiced by nurses.