Gentamicin is a bactericidal aminoglycoside antibiotic, it inhibits the protein synthesis. Gentamicin is active against the majority of aerobic gram-negative bacilli such as Pseudomonas, Klebsiella and Escherichia coli. The gentamicin doses are 3 mg/kg once-daily for preterm newborns < 35 weeks of gestation and 4 mg/kg once-daily for newborns > 35 weeks of gestation. The monitoring of gentamicin serum concentration is recommended when infants are treated for 48 hours or more. The gentamicin peak concentration must be at least 8 times the minimum inhibitory concentration (MIC) to be bactericidal and the gentamicin trough concentration must be < 2 µg/ml to avoid ototoxicity and nephrotoxicity. Once-daily dosing of gentamicin (4 mg/kg), is preferred than twice-daily dose of 2.5 mg/kg gentamicin. A gentamicin loading dose (4 mg/kg), followed by once-daily dosing of 2.5 mg/kg yields safe and target range in neonates. An extended dosing interval of 48-hour (5 mg/kg gentamicin), was compared with twice-daily dose of 2.5 mg/kg gentamicin. Infants in the 48-hour interval and in the twice-daily achieved peak gentamicin concentrations of 9.43 µg/ml and 6.0 µg/ml, respectively, (p<0.001), and trough gentamicin concentrations were 1.08 µg/ml and 1.54 µg/ml, respectively, (p<0.001). The infants born small for gestational age have a reduced gentamicin clearance, and a more prolonged gentamicin half-life than infants born appropriate for gestational age. Patent ductus arteriosus, extracorporeal membrane oxygenation, therapeutic hypothermia, and asphyxia reduce the gentamicin clearance.