BACKGROUND፡ Globally, over 3 million newborn die each year, one million of these attributed to infections. The objective of this study was to determine the etiologies and clinical characteristics of sepsis in neonates admitted to intensive care unit of a tertiary hospital in Ethiopia.METHODS: A longitudinal hospital based cohort study was conducted from April 1 to October 31, 2018 at the neonatal intensive care unit of Jimma Medical Center, southwest Ethiopia. Diagnosis of sepsis was established using the World Health Organization’s case definition. Structured questionnaires and case specific recording formats were used to capture the relevant data. Venous blood and cerebrospinal fluid from neonates suspected to have sepsis were collected.RESULTS: Out of 304 neonates enrolled in the study, 195 (64.1%) had clinical evidence for sepsis, majority (84.1%; 164/195) of them having early onset neonatal sepsis. The three most frequent presenting signs and symptoms were fast breathing (64.6%; 122/195), fever (48.1%; 91/195) and altered feeding (39.0%; 76/195). Etiologic agents were detected from the blood culture of 61.2% (115/195) neonates. Bacterial pathogens contributed for 94.8% (109/115); the rest being fungal etiologies. Coagulase negative staphylococci (25.7%; 28/109), Staphylococcus aureus (22.1%; 24/109) and Klebsiella species (16.5%; 18/109) were the most commonly isolated bacteria.CONCLUSION: Majority of the neonates had early onset neonatal sepsis. The major etiologies isolated in our study markedly deviate from the usual organisms causing neonatal sepsis. Multicentre study and continuous surveillance are essential to tackle the current challenge to reduce neonatal mortality due to sepsis in Ethiopia.KEYWORDS: Neonatal mortality, neonatal sepsis, isolates, blood culture, Ethiopia