Background/Objective: Patients with beta-thalassemia are more susceptible to iron overload and have altered neutrophil function. This study investigated the connections between iron metabolism in neutrophils, neutrophil functionality, and overall iron status in individuals with β-thalassemia and sickle cell anemia. Methods: We recruited 18 patients with β-thalassemia, 5 patients with sickle cell anemia, and 15 healthy controls. Our evaluation included measurements of iron and hepcidin concentrations in the serum, along with an analysis of neutrophil function, specifically their phagocytic and oxidative burst capabilities. In addition, we examined the expression of iron transport proteins in neutrophils. Results: Patients with β-thalassemia showed significant iron overload, reduced neutrophil counts, and decreased oxidative burst activity and phagocytosis. Systemic iron status is inversely correlated with the phagocytic capacity of β-thalassemia neutrophils. Regression analysis indicated a significant association between serum iron level, transferrin iron binding capacity, transferrin saturation, and neutrophil percentage. These findings elucidate the essential role of systemic iron levels in neutrophil efficacy against infections. Furthermore, FPN1B and DMT1A mRNA levels were upregulated, and IRP2 was downregulated in the neutrophils of patients with β-thalassemia major and intermedia compared to controls. Conclusions: Elevated systemic iron levels were associated with reduced neutrophil counts and impaired neutrophil function in patients with β-thalassemia. These findings highlight a critical role of systemic iron overload in neutrophil dysfunction.