Gentamicin, an aminoglycoside antibiotic, is widely used in neonatal intensive care units (NICUs) for its efficacy against Gram-negative pathogens. However, its pharmacokinetics (PK) and pharmacodynamics (PD) are influenced by neonatal physiology, necessitating tailored dosing strategies. This review examines the comparative efficacy and safety of extended-interval dosing (EID) versus traditional dosing (TDD) in premature and term neonates. It also evaluates the role of PK/PD principles, particularly therapeutic drug monitoring (TDM) and the area under the curve to minimum inhibitory concentration (AUC/MIC) ratio, in optimizing gentamicin therapy. EID demonstrates superior efficacy and safety by achieving optimal Cmax/MIC ratios and reducing nephrotoxicity and ototoxicity risks, making it the preferred regimen in most neonatal scenarios. Future integration of advanced pharmacometric models and biomarkers promises further improvement in individualizing gentamicin dosing.