Objective: Sepsis is one of the serious infections of neonatal period. Since signs and symptoms of neonatal sepsis are nonspecific and delayed therapy may cause high mortality, empirical antibiotic therapy is suggested to cover common causative organisms. The etiology of neonatal sepsis may vary from country to country. The aim of this study was to determine bacterial pathogens causing neonatal sepsis in Ahvaz, a southwest province of Iran.Subjects and Methods: During one year period, from December 2004 to November 2005, all neonates with probable diagnosis of neonatal sepsis who were admitted in two major pediatric hospitals of Ahvaz, were included in this study. Two blood samples were obtained from all neonates and cultured in Ethylene Methylene Blue, McConkey and sheep blood agar media. After primary isolation of organisms, complementary diagnostic tests were done for definite diagnosis of pathogens. Results: Of 593 neonates with probable diagnosis of sepsis admitted in the mentioned hospitals, 83 had positive blood cultures. Fifteen cases were excluded because of isolation of unusual organisms, isolation from one sample tube or contamination. Overally, 68 cases (11.3%) had culture-proven sepsis. Of which 20 (29.6%) presented as early–onset sepsis and 48 (70.6%) were late-onset sepsis. The bacterial pathogens encountered in order of frequency were Klebsiella pneumonia (28%), Staphycococcus epidermidis (20.5%), Enterobacter (7%), Escherichia coli (16%), Pseudomonas aeruginosa (15%) and Staphycococcus aureus (1.5%). Group B streptococcus was not isolated from these cases. Most cases of S. epidermedis and Pseudomonas aeruginosa had history of previous admission in other hospitals.Conclusion: The results of this study showed that gram-negative organisms are the most common pathogens causing neonatal sepsis in Ahvaz. This should be considered when choosing appropriate antibiotics for empirical therapy of neonatal sepsis in this area.