Background - Even with advancement in the care provided for patients in Neonatal Intensive Care Units (NICU) and wide spread use of antibiotics, sepsis remains an important cause of high mortality and morbidity. This study was done to determine the Incidence of bacterial isolates. Objective - We aimed to investigate bacterial pathogens causing neonatal sepsis in the neonatal intensive care unit of Hamad Medical Corporation, Doha, Qatar. Materials and methods - Descriptive and retrospective study between August 2006 and June 2008, Neonatal Intensive Care Unit of Hamad Medical Corporation in Doha, Qatar. All neonates with culture-proven sepsis admitted to Neonatal Intensive Care Unit during study period. Results - Out of 2,851 blood culture sent to the laboratory 302 were positive. These cultures were obtained from 176 neonates resulting in sepsis incidence rates of 6.4 cases per 1,000 live births and case-fatality rates of 17%. Gram positive cocci, fungi, and gram negative bacilli made up 66%, 17.8%, and 16.2% of isolates respectively. Conclusion - Gram positive cocci are the major causes of neonatal sepsis in Doha. The high incidence rates of fungal sepsis are associated with increased mortality risk. Good infection control practice together with sensible antibiotic use and on-going surveillance would result in proper neonatal sepsis management, decrease in associated morbidity and mortality.