Background: Nurses have a major role in caring for congenital hypothyroidism (CH) neonates in the pre-analytic phase of the Newborn Screening Program (NSP) as well as at the post-analytic phase. Objectives: To assess performance of nurses working in the pre-analytic phase of the Egyptian NSP for CH at Kafr El-Dawar Health District, El-Behaira Governorate and to assess their knowledge regarding this program before and after implementation of educational sessions. Methods: Cross-sectional and quasi-experimental designs were used to conduct the study in 39 health units, representing all health units in Kafr El-Dawar health district, and included 122 nurses working in these units. Observational checklist was used to assess their screening performance during collection of samples and their adherence to the program guidelines.The Knowledge was assessed before and after the application of an educational program using knowledge assessment sheet. Results: Adherent nurses' performance was observed  at all heath centres (100%) regarding the following items: the use of appropriate filter paper in collecting the samples; the accuracy in writing cards information; disinfection of the heel; putting the used lancet in the safety box and sending sampling cards to lab in the same day  (5 items out of 20 items). On the other hand, washing hands before every sampling was not done in 100% of observations. The lancet was used in the appropriate site in nearly two thirds of observations (74.6%).The sample cards were kept in its special rack in nearly 73.8% of observations. The screening cards were put in its envelope after drying in 81.1% of observations. Health education of mothers about CH in general and CH complications in particular were done with only 57.4% and 28.7% of cases respectively. It was not done about the symptoms of CH in 100% of the observations. There was a statistically significant improvement in nurses' Knowledge after the educational program. Recommendations: Continuous education programs should be mandatory for all nurses providing pre-analytical phase services of NSP for CH. In addition, adequate supervision, guidance and regular feedback by head nurses as well as availability of clinical protocols and guidelines are greatly needed to improve nurse's performance. To facilitate implementing the guidelines by nurses, adequate infection control materials should be available in units providing the service.