Abstract Background: Early awareness of pregnant women about neonatal jaundice (NNJ) has an essential role in preventing severe hyperbilirubinemia and its dangerous complications. Aim: the study aimed to assess the level of knowledge and attitude of pregnant women regarding NNJ and to evaluate the effect of an educational program for neonatal jaundice among the target population. Subject and methods: three hundred pregnant women attended the Antenatal Clinic at Women Health Hospital in Assiut University were divided into 150 pregnant women as study and 150 as control groups. Quasi-experimental research design was applied. Tool: Using a structured interview questionnaire including five parts; personal data, obstetric, medical and family history, assessment of knowledge, attitude toward neonatal jaundice, and neonatal follow up. Results: There was a statistically significance difference between level of knowledge and attitude in pre- vs. post-test in the study group compared with the control group and there was statistical evidence of neonatal outcomes for improving women's breast feeding and decrease number of jaundice days. Conclusion: This study revealed that pregnant women had inadequate knowledge regarding NNJ and their attitude was negative while the application of an educational program has led to significant improvement in the level of women's knowledge and attitude in the study group, which was reflected on the newborn positively. Recommendations: Implementing a routine health education program for pregnant women about NNJ and raising awareness of the relatives as they were identified as the main source of knowledge.