Introduction: Myeloperoxidase (MPO) is a heme enzyme of neutrophils (polymorphonuclear leukocytes) (PMNLs) azurophilic granules with a strong oxidative activity. MPO has been demonstrated to be a major pathway for O2-dependent microbicidal activity. Together with the membranous NADPH oxidase, MPO is involved in the formation of reactive oxygen species (ROS) and oxidation of biological material. Objective: This cross-sectional study aimed to evaluate the diagnostic utilities of MPO enzyme activity for diagnosis of neonatal sepsis. Subjects and methods: The study was performed on neonates admitted in the Neonatal Intensive Care Unit (NICU) of Cairo University Hospital (Kasr El Aini) and New Children's Teaching Hospitals (El-Monira) between July 2012 and January 2013; in whom infection was confirmed by positive blood culture. A total of 84 neonates were studied; 41 were culture proven neonatal sepsis and 43 healthy neonates served as controls. Complete blood count with differential leucocytic count, C-reactive protein (CRP) quantitative assay and venous blood gases were done in cases group while MPO enzyme activity measured by ELISA and compared between both groups. Results: MPO enzyme activity levels were statistically higher in case group compared to control group (P-value < 0.001). There was no statistically significant difference whither the infected was pre-term (PT) or full term (FT). MPO enzyme activity levels were statistically higher in case with sepsis without septic shock compared to cases with septic shock with P-value < 0.001 .MPO enzyme activity showed positive moderate correlations with TLC, absolute neutrophil count and absolute segmented cells count; while positive weak correlation with absolute band cells count. MPO assay (using the cutoff value of 146.5ng/ml) had a sensitivity of 80% and specificity of 83.9% with a PPV of 92.9%, NPV of 61.5% and accuracy of 82.9%. Conclusions: MPO enzyme activity was proven to increase significantly in neonates with sepsis. MPO enzyme activity not affected by gestational age but can be affected by deterioration of the case to septic shock. Early diagnosis of NS cannot rely on a single laboratory test and clinical decision remains to have the upper hand in the diagnosis.