Because of the high susceptibility to infections, antibiotics are the most widely used drugs in newborns. The result of antibiotic use, however, may be strongly influenced by the peculiar physiology of the neonate, characterized by the delicate process of adaptation from intra- to extra-uterine life. Additional important factors that may affect antibiotic therapy are gestational age, birth weight, the intrauterine growth restriction, chronological age and, especially, the kidney and liver function immaturity. Dosing, timing and route of administration must, therefore, take in careful consideration the neonatal variability of bioavailability, distribution, metabolism, biotransformation, and excretion. The fine adjustment of dosing and duration of therapy should be based on pharmacokinetic and pharmacodynamic parameters. In spite of significant variations of sepsis etiology, the best initial empiric therapy of a suspected systemic infection still remains, as several years ago, the association of ampicillin and gentamicin. Other very effective and useful antibiotics, as cephalosporins, carbanepems or glycopeptides should be administered judiciously to infants, following the recommendations of a restricted use, to obtain maximal efficacy and minimal toxicity. Finally, because of their peculiar features, macrolide antibiotics have recently been proposed for different indications than the antibacterial activity. Use of oral erythromycin for the treatment of gastrointestinal dysmotility in preterm infants could reduce the incidence of parenteral nutrition-associated cholestasis by almost 50%, while azithromycin because of the combined antibiotic and anti-inflammatory effects, has been successfully used in a pilot study in the extremely low birth weight infant for the prevention of bronchopulmonary dysplasia.