Background: Neonatal Sepsis (NS) is a major health risk for newborns, particularly those bornpreterm, and is a primary contributor to morbidity. Research has identified Macrophage Migration Inhibitory Factor (MIF) as a key player in the devel-opment of sepsis and other immunerelated diseases. Aim of Study: The current research aimed to quantify MIF-concentrations in neonates with early and late-onset sepsis and correlate its levels with other key clinical and laboratory indi-cators in comparison to non-septic neonates. Patients and Methods: Thiscross-sectional research was conducted from March to September 2023 and involved 32 neonates with Early-Onset (EONS, n=16) and Late-Onset (LONS, n=16) neonatal sepsis, confirmed by positive blood cultures, compared to another 32 non-septic neonates (con-trols), matched for age and gender. All neonates underwent ba-sic laboratory tests, and MIF levels were assessed using ELISA. Results: The mean levels of MIF were significantly high-er in both early-onset (169.11±51.88ng/ml) and late-onset (150.6±57.43ng/ml) neonatal sepsis groups, in comparison with the non-septic group (41.97±17.5ng/ml, p-value=0.000). The two groups with sepsisexhibited comparable MIF values. A cutoffvalue >66.7ng/ml for MIF levels was determined to dis-criminate between septic and non-septic neonates with (100% sensitivity; 96.87% specificity; positive predictive value: 97%; negative predictive value: 100%; AUC: 0.997). Furthermore, MIF levels were negatively correlated with absolute lympho-cytic count, PH level in early-onset and random blood glucose in late-onset neonatal sepsis groups. Conclusion: MIF could be an excellent and early diagnos-tic marker for NS even in preterm neonates. Further studies are needed to correlate MIF levels with severity of neonatal sep-sis and compare the diagnostic and prognostic utility of Mac-rophage Migration Inhibitory Factor with other inflammatory biomarkers, like procalcitonin and C-Reactive Protein (CRP).