The aim of this study was to investigate the effect of education program on nurses’ knowledge and practice in pediatric HD unit evidence-based practice (EBP) guidelines. Participants and methods A quasiexperimental design was utilized. The study was conducted in the Pediatric Hemodialysis Unit at Benha University Hospital and the Hemodialysis Unit at Benha Fever Hospital. A convenient sample of 40 nurses from the previously mentioned setting was included in the study. Two tools were used: a questionnaire sheet for assessing nurses’ personal data and knowledge about CRBSIs and EBP guidelines, and an observational checklist to assess nurses’ practice toward care of pediatric HD catheter. Results The study results showed that there was a highly statistically significant improvement in total knowledge level of nurses immediately after implementation and 6 months after implementation of education program guidelines. Moreover, there was a highly statistically significant relation in nurses’ practice and their educational level, occupation, years of experience, and attendance of training course, before implementation/immediately after implementation and 6 months after implementation. Conclusion The current study concluded that the nurses who received evidence-based guidelines educational program as regards central vascular catheter maintenance and care showed high score level in nurses’ knowledge and practice after implementation of the program than that before recommendation. The study recommended that training and continuing education of the EBP guidelines for staff nurses are important to improve care provided in the pediatric hemodialysis units.