Aim: Early-onset neonatal sepsis (EONS) occurring within the first 72 hours after birth is a common, life-threatening disease in neonatal intensive care units (NICUs). The limited accuracy of diagnostic tools makes the diagnosis of EONS difficult, and the quest for new markers remains open. Automated hematology analyzer-derived neutrophil cell population data (N-CPD) have been identified as a potential marker of neonatal sepsis, but their role for EONS has not been elucidated yet. Our aim is to explore the role of automated hematology analyzer-derived N-CPD as a marker of EONS. Methods: We prospectively evaluated a cohort of 289 neonates admitted to the NICU with clinical signs of sepsis, and checked if N-CPD from the Beckman Coulter UniCel DxH 800 device could help identify those who would develop culture-proven EONS. Clinical characteristics, sepsis markers, blood culture results, and N-CPD were recorded. The diagnostic accuracy of N-CPD was tested using receiver-operator curves (ROCs). Results: Receiver-operator curves of the standard deviation of neutrophil volume (SD-V) showed moderate accuracy in identifying EONS (AUC 0.74), with a high negative predictive value (NPV 98.6%) for cut-off values >21.76 arbitrary units. Accuracy was higher with VCS at 12–48 hours of life (AUC 0.8). Standard deviation of neutrophil volume accuracy was independent from gestational age (GA), birth weight, and timing of test execution (OR 1.14, p = 0.002; AUC 0.71). Conclusion and significance: Our study confirms the role of N-VCS in the diagnostic workup for EONS. High NPV values may be useful as they suggest a role as an adjunctive marker useful for ruling-out EONS and support early empirical antibiotic withdrawal.