Background: Jaundice is one of the most common conditions confronting neonatologists daily. Phototherapy is generally regarded as a safe method for treating hyperbilirubinemia, but it may also lead to undesired effects, one of these effects that it can affect the function of the immune system of the newborn. Aim of the work: The aim of the work was to study the effect of phototherapy on the serum level of tumor necrosis factor-alpha [TNF-α] in neonates. Patients and methods: A total of 35 cases of term neonates with Neonatal Jaundice and indirect hyperbilirubinemia high to the level that need phototherapy for 72hrs or more according to the guidelines of the American Academy of Pediatrics were included in this study, and 15 healthy matched newborns were selected as controls. TNF-α was measured before exposure and after 72 hours phototherapy and from control group at the time of examination. Serum levels of TNF alpha were measured using enzyme linked immunosorbent assay kits. Results: The results showed that there was no statistically significant in patient group before phototherapy in comparison to control group as regards TNF-α, patient demographics or laboratory data. On the other side, serum TNF-α levels significantly increased after exposure to phototherapy for 72 hours when compared to values before phototherapy (151.49±61.97 vs 61.36±31.96 respectively), indicating the influence of phototherapy on serum level of TNF-α. Conclusion: The results demonstrated increased serum TNF-α level after 72 hours may affect the immune system in neonates.