Bacterial infections are an important cause of mortality and morbidity in newborns. The main risk factors include low birth weight and prematurity. The study identified the most common bacterial pathogens causing neonatal infections including their resistance to antibiotics in the Neonatal Department of the University Hospital Olomouc. Additionally, the cut-off for distinguishing early- from late-onset neonatal infections was assessed. The results of this study show that a cut-off value of 72 h after birth is more suitable. Only in case of early-onset infections arising within 72 h of birth, initial antibiotic therapy based on gentamicin with ampicillin or amoxicillin/clavulanic acid may be recommended. It has been established that with the 72-h cut-off, late-onset infections caused by bacteria more resistant to antibiotics may be detected more frequently, a finding that is absolutely crucial for antibiotic treatment strategy.