The incidence of infections is higher in the neonatal period than at any time of life. The basic treatment of infants with infection has not changed substantially over the last years. Antibiotics (with or without supportive care) are one of the most valuable resources in managing sick newborn babies. Early-onset (ascending or transplacental) or late-onset (hospital acquired) infections present different chronology, epidemiology, physiology and outcome. Some classes of antibiotics are frequently used in the neonatal period: penicillins, cephalosporins, aminoglycosides, glycopeptides, monobactams, carbapenems. Other classes of antibiotics (chloramphenicol, cotrimoxazole, macrolides, clindamycin, rifampicin and metronidazole) are rarely used. Due to emergence of resistant bacterial strains in Neonatal Intensive Care Units (NICU), other classes of antibiotics such as quinolones and linezolid will probably increase their therapeutic role in the future. Although new formulations have been developed for treatment of fungal infections in infants, amphotericin B remains first-line treatment for systemic Candida infection. Prophylactic antibiotic therapy is almost always undesirable. Challenges from pathogens and antibiotic resistance in the NICU may warrant modification of traditional antibiotic regimens. Knowledge of local flora and practical application of different antibiotic characteristics are key to an effective and safe utilization of antibiotics and antifungals in critical newborns admitted to the NICU, and especially in very low birth weight infants.