Background: While surfactant administration therapy significantly reduces respiratory distress syndrome mortality among preterm infants. Studies indicate persistent variations in nursing knowledge and practices across neonatal units, including inconsistent adherence to administration protocols, gaps in monitoring post- surfactant administration therapy complications, and limited training on emerging minimally invasive techniques. Aim: to evaluate the effect of guidelines about surfactant administration for preterm infants on nurses’ knowledge and practices. Methods: A quasi-experimental design has been conducted at two neonatal intensive care units of El Manial University Hospital (Kaser Al Aini) and Cairo University Children’s Hospital (El Monira) from December 2024 to May 2025. A convenient sample of fifty bedside nurses who are working in previous mentioned setting was included using two tools: structured questionnaire and observation checklist regarding surfactant administration. Results: the current study findings revealed that nurses' level and total mean scores knowledge and practice of surfactant administration significantly improved following intervention of guidelines compared to pre intervention. There was moderate positive correlation between nurses’ knowledge and practice scores across all study phases and educational qualification only demonstrated a strong positive correlation with both knowledge and practice scores. Conclusion: Nurses who receive guidelines had a higher mean post- test scores of surfactant administration knowledge and practices compared to pretest. The findings strongly support the effectiveness of the guidelines in improving neonatal nurses’ knowledge and practices regarding surfactant administration for preterm infants Recommendation: Implementing periodical instructional guideline for nurses working in neonatal intensive care units regarding surfactant administration for preterm infants.