Background: Neonatal sepsis is an important cause of neonatal mortality and morbidity worldwide. The common causative bacteria are Group B Streptococci, Escherichia coli, Listeria monocytogenes, Staphylococcus, Enterococci, Klebsiella, and Enterobacter. The risk factors are premature rupture of membrane, meconium-staining, foul-smelling amniotic fluid, low birth weight, prematurity, low Apgar score, mode of deliveries, and mechanical ventilation. The treatment for neonatal sepsis is a beta-lactam antibiotic combined with an aminoglycoside. The aim of the study was to find the incidence and factors associated with culture-positive neonatal sepsis, and common bacterial isolates. Methods: A hospital-based retrospective cross-sectional study was conducted among 385 neonates admitted with neonatal sepsis at Narayani Central Hospital, Birgunj from July 2021 to July 2022. Ethical approval was taken from the Institutional Review Committee, Nepalese Army Institute of Health Sciences, Nepal (Reference no. 669/ 2022). A convenient sampling was followed. Data regarding different clinical factors were collected in a structured questionnaire. The data was entered and analyzed through SPSS version 22.0 using a binary regression model. Results: The study revealed the mean age, weight, and gestational age of neonates were 2.79kg±0.55kg, 6.19±3.87 days, and 37.64±1.27 weeks respectively. Out of the total cases, 26.5% (102) were culture-positive neonatal sepsis. Staph aureus was the predominant pathogen (40, 39.2%) followed by Klebsiella (25, 24.5%) and Acinetobacter (16, 15.7%). The Premature rupture of membrane (OR=4.32, 95% CI: 2.20–8.47) and Mothers with foul-smelling amniotic fluid (OR=3.03, 95% CI: 1.32–6.92) are statistically associated with culture-positive neonatal sepsis. Conclusion: The factors responsible for the high burden of neonatal sepsis such as premature rupture of membrane, foul-smelling amniotic fluid, prematurity, low birth weight, unhygienic home delivery practices, can be avoided dramatically. The role of pediatricians, nurses, hospital administrations and general awareness among mothers about hygienic practices during parturition are essential to minimize neonatal mortality and morbidity.