The work nature of nurses in pediatric oncology unit necessitates ethical care more than routine care. Therefore, nurses require knowledge about nursing ethics in this area to guide their practice in a competent manner. Aim of this study was to evaluate the effect of guideline education program about nursing ethics on nurses’ knowledge and practice in pediatric oncology unit. Setting: The study was conducted in Oncology/Hematology Unit at Benha Specialized Pediatric Hospital. Research design: Quasi-experimental design was utilized. Sample: A purposive sample of 32 nurses who worked at the previously mentioned setting. Tools: Data were collected through two main tools; (I) a structured interview ethical responsibilities questionnaire sheet, it was consisted of two parts; personal characteristics of the studied nurses and nurses’ knowledge about ethical behavior in pediatric oncology unit. (II) Observational checklist for assessing nurses’ practice regarding application of ethical principles toward dealing with children in pediatric oncology unit. Results: of this study showed that, mean age of the studied nurses was 25.97 ± 4.99 years. There were highly statistical significant differences in relation to nurses’ knowledge and practice regarding their ethical responsibilities in pediatric oncology unit at pre and post-program intervention. Conclusion: this study concluded that nurses’ knowledge and practice toward their ethical responsibilities in pediatric oncology unit was improved after implementation of the guideline education program. Recommendation: Encourage nurses to attend training courses about their ethical responsibilities toward children in oncology unit