Background: Antibiotic stewardship is the optimum selection of antimicrobial therapy that results in the best clinical outcome for the management/ prevention of infection, with slight toxicity and minimal influence on subsequent resistance. The aim of the study was to assess the effect of an educational program on nurses' performance regarding antibiotic stewardship among high-risk neonates. Design: A quasi-experimental (pre & post-test) design used. Settings: This study was performed at Neonatal Intensive Care Units and Neonatal Surgical Intensive Care Unit of Benha Specialized Pediatric Hospital in Benha city. Sample: A purposive sample of 80 nurses and a purposive sample of 74 high-risk neonates at the previously mentioned settings. Tools of data collection: Four data collection tools were utilized, a structured interviewing questionnaire, nurses’ practices observational checklists, nurses’ attitudes sheet, and High-risk Neonates' Medical Record. Results: The current study demonstrated that most studied nurses (88.8%, 83.8% respectively) had a satisfactory level of knowledge and proficient level of practices after applying the educational program as compared to (8.8%, 17.5% respectively) before implementation. Conclusion: Based on the findings of the current study, studied- nurses had a satisfactory level of understanding, attitudes, and proficient level of practices after implementation of the educational program as compared to pre-program implementation with a statistically important improvement. Recommendations: The study suggest the application of traditional and online in-service training in NICUs for nurses with consistent revising of their knowledge and practices concerning antibiotic stewardship program for high-risk neonates in all NICUs.