Background/Objectives: Busulfan is a key component of myeloablative conditioning regimens in hematopoietic stem cell transplantation (HSCT) for pediatric patients with acute myeloid leukemia, solid tumors, and certain non-malignant diseases. This study compares the clinical outcomes of once-daily (BU1) versus four-times-daily (BU4) busulfan dosing regimens in pediatric HSCT recipients. Methods: A retrospective analysis was conducted on 70 pediatric patients who underwent HSCT at Hadassah Medical Center between June 2018 and October 2023. Thirty-five patients received the BU4 regimen, and 35 received BU1. The primary endpoint was 100-day event-free survival (EFS). Results: There was no statistically significant difference in 100-day event-free survival between the BU1 group (88.6%) and the BU4 group (85.7%; p = 0.768). Similarly, no significant differences were found in time to neutrophil engraftment (p = 0.251) or platelet engraftment (p = 0.688). Sinusoidal obstruction syndrome (SOS) occurred in 17.1% of patients in each group. No significant differences were observed in the increase in liver enzyme levels (p = 1.0). The incidence of acute graft-versus-host disease was comparable between the groups (41.9% for BU1 vs. 40.0% for BU4; p = 0.878). Conclusions: Once-daily and four-times-daily busulfan regimens demonstrated comparable clinical outcomes in terms of efficacy and adverse events. Further prospective studies are needed to validate these findings.