Background <p>Antimicrobial resistance represents a great global concern and initiating an antimicrobial stewardship program is one of the main efforts to control antimicrobial resistance through optimizing antimicrobial utilization. Antibiotics are used extensively and inappropriately in neonatal intensive care units (NICUs). The present study aimed to assess the clinical impact of the antibiotics stewardship program (ASP) in the NICU.</p> Methods <p>The study was conducted in two phases at the NICU, Assiut University Children’s Hospital, where 1200 patients were enrolled from January 2019 to June 2020. The pre-ASP phase included making NICU-specific antibiograms, choosing the antibiotic use evaluation measures, conducting antibiotic use evaluations, and designing the ASP. The ASP intervention phase included the implementation of ASP, which involved modifying the neonatal sepsis treatment protocol according to the local antibiotic susceptibility patterns and measuring its clinical outcomes. Categorical data were tested with Pearson’s chi-squared test and Fisher’s exact test. Numerical data were tested with the Wilcoxon rank sum test.</p> Results <p>A total of 603 and 597 patients were enrolled in the pre-ASP and intervention-ASP phases, respectively. The ASP intervention phase showed a significant increase in the number of C-reactive protein tests [1(1–2)vs 2(1–3),<i>p</i> = 0.001], microbiological cultures per patient [0(0–1) vs. 1(0–1),<i>p</i> = 0.001], the number of patients taking definitive therapy [15.1% vs. 20.1%,<i>p</i> = 0.023], and the number of pharmacist interventions per patient [1(1–1) vs. 1(1–2),<i>p</i> = 0.001]. The duration of the antibiotic course [9(5–16) vs. 8(4–15),<i>p</i> = 0.04] and the number of patients taking empirical therapy [38.6% vs. 30.7%,<i>p</i> = 0.004] were significantly decreased in the ASP intervention phase. The prescribing rates of antibiotics and their consumption in terms of length of therapy were changed according to the NICU-specific antibiogram; however, days of therapy were unchanged in the ASP intervention phase. There was a significant reduction in the 14-day [46% vs. 28%,<i>p</i> = 0.03] and 28-day [64% vs. 43%,<i>p</i> = 0.022] mortality of patients with late-onset sepsis after modifying the neonatal sepsis treatment protocol according to the local antibiotic susceptibility patterns in the ASP intervention phase.</p> Conclusion <p>ASP implementation was successful in improving antibiotic prescribing and modifying the neonatal sepsis treatment protocol according to the local antibiotic susceptibility patterns, which resulted in reduced 14- and 28-day mortality.</p> Trial registration <p>Clinical Impact of an Antibiotic Stewardship Program in a Neonatal Intensive Care Unit, Registration number NCT04039152. Registered 31 July 2019 - Retrospectively registered. <a href="https://classic.clinicaltrials.gov/ct2/show/NCT04039152">https://classic.clinicaltrials.gov/ct2/show/NCT04039152</a>.</p>