Background: Neonatal sepsis is a systemic inflammatory response syndrome that is secondary to infection. It is a major cause of neonatal mortality in the world, particularly in developing countries. A definitive diagnosis requires the isolation of pathogens from a normally sterile body site, including blood, cerebrospinal fluid and urine. Empirical antibiotic therapy is based on the physician's knowledge of the anticipated bacterial species and their expected antibiotic susceptibilities. Objectives: The aim of this study was to determine the prevalence and evaluate the antimicrobial susceptibility patterns of bacterial infections at a neonatal unit. Patients and Methods:This study was conducted at the neonatal intensive care unit and neonatal ward of Hakim hospital, Neyshabour, Iran. Blood, cerebrospinal fluid (CSF) and urine specimens were collected before institution of empirical antibiotic therapy. Antibiotic resistance pattern of the isolates was studied by the disc diffusion technique. Results: Coagulase-negative staphylococci (CoNS) were the most prevalent pathogens isolated from blood specimens in early and late-onset disease. Escherichia coli and Klebsiella were the most causative pathogens in early and late-onset urinary tract infections. They had high resistance to our empirical antibiotic regimens. Prevalence of bacterial meningitis was low in our study. Conclusions: Due to the increasing resistance of pathogens to usual empirical antibiotics, it is reasonable to stress upon preventive measures, so that a minimum number of neonates develop sepsis.