Background:Sepsis is a commonly encountered and potentially life-threatening problem in neonatal intensive care units, blood culture of neonatal sepsis helps in either optimizing treatment or terminating antibiotics.Materials and Methods:We determined the causative agent, time to positivity (TTP), and antibiogram of neonatal blood cultures collected in a tertiary care center, to investigate difference between early- and late-onset neonatal sepsis and to establish the time at which a blood culture could safely be considered negative, using the BacT/ALERT® 3D 60. A total of 826 clinically suspected neonates suffering from sepsis and admitted to a neonatal intensive care unit of a tertiary care hospital, Alexandria, Egypt were included in this study.Results:Eighty-five (10.29%) showed positive results. The overall TTP median was 21.1 h. Out of the 85 positive cultures, 57 (67.06%) were Gram-positive, 15 (17.65%) were Gram-negative, and 13 (15.29%) were fungi (all Candida). Coagulase-negative staphylococci were the predominant organism (41.18%). All the Gram-positive pathogenic isolates were sensitive to vancomycin and tigecycline. Among the Gram-negative isolates, maximum antibiotic sensitivity was observed for levofloxacin.Conclusion:We conclude that more than 3 days of incubation may not be required when using the BacT/ALERT® 3D 60 system.