Introduction: Despite the fact that pre-screening education for mothers is widely acknowledged as important, mothers still have little information about neonatal screening. Aim of the study: this study aimed to assess the knowledge and attitude of Egyptian mothers regarding neonatal screening tests for metabolic disorders and hearing loss. Design: to conduct this study, a descriptive cross-sectional research approach was selected. Setting: The study was carried out at Neonatal Unit and Pediatric Outpatient Clinics of Tanta Main University Hospital, El-Menshawy Hospital and El-Mabara Hospital. Subjects: A convenience sampling of 1500 mothers. Tools: Three tools were used. Tool I: Mothers' sociodemographic characteristics. Tool II: Mothers' knowledge about neonatal screening tests. Tool III: Mothers' attitude towards neonatal screening tests. Results: nearly two thirds of mothers showed sufficient level of general knowledge, while the majority of them had insufficient level of total specific knowledge about neonatal screening tests. More than three-quarters of mothers showed a positive attitude regarding neonatal screening tests. Conclusion: mothers had sufficient level of general knowledge and insufficient level of specific knowledge with positive attitude about screening tests. Recommendations: develop an educational program for mothers regarding neonatal screening at maternity and child health centers.