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Background: Neonatal sepsis is a significant cause of morbidity and mortality in low- income countries. Neonatal sepsis is classified as early-onset neonatal sepsis (EONS) and late-onset neonatal sepsis (LONS). Etiologies responsible for EONS are mostly acquired vertically from the mother during or before birth with the possibility of prevention. The burden and etiology of neonatal sepsis is not uniform across the globe with huge disparities based on the income level of the countries. This study aimed to determine neonatal sepsis trends, prevalence, and etiologies at Hawassa University Comprehensive Specialised Hospital (HUCSH). Methods: A hospital-based retrospective cross-sectional study was conducted among newborns aged 0 to 90 days who were admitted to the HUCSH from January 2019 to July 2023. Patient-related information and the culture results were obtained from HUCSH microbiology laboratory registration book. Data analysis was performed using SPSS version 25 software. Results: Out of 2364 newborns suspected of having sepsis, 56% (95% CI: 54−58%) had culture-confirmed sepsis. When excluding Coagulase Negative Staphylococcus (CONS), the prevalence of culture-confirmed neonatal sepsis was 36.9%. The highest numbers of culture-confirmed cases was observed in 2021. The predominant bacteria identified were Coagulase Negative Staphylococcus (CONS) (34.1%), Klebsiella pneumoniae (12.9%), and Enterococcus (10.6%). Among culture-confirmed neonatal sepsis, 59.9% and 40.1% of cases were EONS and LONS, respectively. Coagulase Negative Staphylococcus and Enterococcus were the major bacteria found in both EONS and LONS while. Klebsiella pneumoniae was the second most common bacteria among newborns with EONS following CONS. Conclusions: The prevalence of culture-confirmed neonatal sepsis was relatively high in the study area. Early-onset neonatal sepsis was consistently more prevalent than LONS. The predominant etiologies of neonatal sepsis excluding CONS were K. pneumoniae, Enterococcus, Enterobacter agglomerans, Acinetobacter species, and Staphylococcus aureus. Among newborns with EONS, the predominant bacteria were K. pneumoniae, Enterococcus, Enterobacter agglomerans, and Acinetobacter species. [ABSTRACT FROM AUTHOR]