Background: Surfactant Replacement Therapy (SRT) is part of the core treatment strategy for respiratory distress syndrome, which prevent collapse of alveoli, improving survival and reducing respiratory morbidities. Evidence-based nursing guideline increases the quality of neonatal care and closes the gap between research outcomes and practice. Aim of the study was to assess the effect of evidence-based guidelines on nurses' performance regarding care of high-risk neonates undergoing SRT. Design: a quasi–experimental research design was utilized to conduct this study. Setting: This study was conducted in Neonatal Intensive Care Unit (NICU) at Benha University Hospital. Sample: A convenient sample of all available nurses (64 nurses) who were responsible for providing care to high-risk neonates and purposive sample of high-risk neonates (30 neonates) who receive SRT and admitted to NICUs. Tools of data collection: Two tools were used: Tool (I): A structured interviewing questionnaire sheet to assess characteristics of the studied subjects and nurses' knowledge regarding care of high-risk neonates undergoing SRT. Tool (II): Observational checklists to assess nurses’ practices regarding care of high-risk neonates undergoing SRT. Results: The majority of the studied nurses had satisfactory total level of knowledge in post-implementation of evidence-based guidelines and the majority of the studied nurses have competent practice in post-implementation of evidence-based guidelines. Conclusion: The evidence-based guidelines was effective in improving nurses’ knowledge and practices regarding care of high-risk neonates undergoing SRT post-implementation of guidelines. Additionally, there was a positive correlation between nurses’ total knowledge and practices. Recommendation: Enhancing nurses’ knowledge and practices regarding care of high-risk neonates undergoing surfactant replacement therapy by encouraging them to attend periodical training courses.