Abstract: Bacterial sepsis is one of the most common causes of significant mortality and morbidity in neonates. The researchers analyzed bacterial isolates and their antibiotic susceptibilities for cases of septicemia in a Neonatal Intensive Care Unit (NICU) of a teaching hospital in Tehran, Iran. During a 36 months period, the incidence of bacteremia and the causing organisms and mortality of sepsis as well as antibiotic susceptibility were investigated. Neonatal Late-Onset Sepsis (LOS) was defined as clinical signs suggestive of infection with a positive Blood Culture (B/C) after 72 h of birth. About 909 neonates were admitted to the NICU. A total of 9.13% of neonates (83/909) had at least one positive B/C after 72 h of birth. The vast majority (56.6%) of sepsis were caused by Gram-negative organism. Gram-positive pathogens accounted for 41% infections. The most common cause of late-onset sepsis was Klebsiela p. (31%) and followed by Staph aureus (18.1%). In this study, the researchers have observed that the old empiric therapy with cephalothine plus Amikacin for suspected late-onset sepsis seems ineffective. Now, however may be the best choice regimen is the combination vancomycin plus amikacin and the vancomycin plus imipenem for the severe ill patients.