Background: Zinc salts can reduce phototherapy duration by precipitating unconjugated bilirubin in the intestine however, zinc toxicity is an issue that must be considered. Theoretically, bilirubin reduction by phototherapy may increase serum zinc levels, making additional zinc supplementation, which cause zinc toxicity. Objectives: To evaluate serum zinc level alterations before and after phototherapy in hyperbilirubinemic newborns. Materials and Methods: A prospective cross sectional case-control study was performed at Neonatal Intensive Care Unit (NICU), Al-Azhar University Hospital, Assiut. 150 newborns aged between 2-7 days divided into 50 healthy neonates without hyperbilirubinemia (control group) and 100 neonates with physiological jaundice who required phototherapy (case group). Participants were treated according to American Academy of Pediatrics (AAP) criteria. Serum zinc level was measured before and after phototherapy. Results: The mean age of onset was approximately 2 days, while the majority of patients were females and delivered with cesarean section. The age at admission was 3.09 ± 0.57 days. In regard to our primary outcomes, serum bilirubin level was decreased significantly after phototherapy to reach 8.47 ± 1.36 mg/dl (p < 0.001). Serum zinc level was increased significantly after phototherapy to reach 75.45 ± 14.94 mcg/mL (p < 0.001). The comparison between control and study groups regarding serum zinc level showed that there was highly statistically significant difference being higher in case group compared to control group (p < 0.001). Phototherapy was associated with a significant increase in serum zinc level in neonates. Conclusions: Phototherapy increases serum zinc level by reducing bilirubin level so additional supplementation of this element can lead potentially zinc toxicity.