Background: The main course of treatment for respiratory distress syndrome includes surfactant replacement therapy (SRT), which prevents alveolar collapse, boosts survival, and lowers respiratory morbidities. Evidence-based nursing guidelines improve neonatal care and bridge the gap between research findings and clinical 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 guideline, there was an improvement in total nurses practices post-implementation of guideline compared to pre-implementation. Conclusion: The evidence-based guideline was effective in improving nurses' knowledge and practices regarding care of high-risk neonates undergoing SRT post- implementation of guideline. Recommendation: Enhancing nurses' knowledge and practices regarding care of high-risk neonates undergoing surfactant replacement therapy by encouraging them to attend periodical training courses.