Background: Antibiotic overuse in Neonatal Intensive Care Units (NICUs) is a major contributor to antimicrobial resistance and adverse neonatal outcomes. This study aims to evaluate baseline antibiotic utilization (AU), identify factors influencing variability, and assess the impact of neonatal characteristics and sepsis incidence. Methods: A multicenter retrospective analysis examined AU in seven NICUs from 2019 to 2023, involving 25,532 neonatal admissions during national antibiotic stewardship program implementation. Data encompassed neonatal clinical parameters, sepsis incidence, and AU metrics, including days of therapy (DOT) per 1000 patient-days. Statistical analyses included correlation assessments and multivariate regression to identify determinants of antibiotic use. Results: Overall, 43.8% of neonates received antimicrobials, with individual NICUs ranging from 24% to 73% (p < 0.001). Antimicrobial-exposed neonates had a mean gestational age of 35.1 weeks [SD ± 4.4] and a mean birth weight of 2360 g [SD ± 970]. Antimicrobial-exposed neonates were generally more premature [35.1 (±4.4) weeks vs. 37.5 (±2.5) weeks (p < 0.001)] and had lower mean birth weight [2360 g (±971) vs. 2817 g (±686) (p < 0.001)] compared to those not exposed to antimicrobials. Total antimicrobial days varied markedly (8761 to 37,683 days), with DOT per 1000 patient-days ranging from 322 to 1031. Antimicrobial use for culture-negative sepsis varied widely among centers, from 23% to 73%. Antimicrobial-exposed neonates had higher all-cause mortality compared to those who did not [(7.5% vs. 3.2%), (p < 0.001)]. Multivariate analysis revealed individual NICU practice patterns remained significant predictors after adjusting for neonatal characteristics. Conclusions: Neonatal antimicrobial use varied significantly among NICUs, driven primarily by institutional practices rather than neonatal demographics. These findings provide nationally representative baseline data to inform neonatal antimicrobial stewardship interventions and offer transferable lessons for other countries seeking to optimize antibiotic use in NICUs amid rising global antimicrobial resistance.