ABSTRACT Background: Sepsis is a life threatening condition that occurs during host-pathogen interaction results damage in tissue or organs. It is the third most cause of neonatal death and kills one million neonates every year globally. Drug resistance is a major cause of clinical unresponsiveness to treatment and rapid progress of sepsis. Objective: The aim of this study was to assess bacterial profile, antimicrobial susceptibility pattern and factors associated with neonatal sepsis in Felege Hiwot Referral Hospital (FHRH). Methods: Hospital based cross sectional study was conducted in FHRH, from April 2018 to July 2018. A total of 412 neonates were recruited in the study. Demographic and clinical data were collected using pre-tested structured questionnaire and medical records. Blood sample was collected aseptically, directly inoculated into trypton soya broth and incubated up to 7 days at 37oc. Bacterial identification and susceptibility test were done following the standard microbiological techniques. Data was analyzed by using SPSS version 23. Logistic regressions were used to see the association between independent and dependent variables. Odds Ratios, p-value and their 95% confidence intervals were calculated and the result was considered statistically significant when a P-value was less than or equal to 0.05. Results: Out of 412 neonates enrolled, 41.26% (170/412) were positive for bacteriology blood culture. From the bacterial isolates K. pneumoniae, 28.2% (48/170) was the predominant bacterial isolates followed by S.aureus, 24.7 (42/170) and E. coli, 14.1% (24/170). Majority of isolates developed resistant to ampicillin and penicillin. The overall multidrug resistance was observed in 78.2% (133/170). Preterm (<37week) [AOR = 2.049; 95% CI (1.151, 3.647)], Low birth weight (<2500 gram) [AOR =2.357; 95% CI (1.352, 4.109)], Prolonged rupture of membrane (>18hours) [AOR = 4.282; 95% CI (1.615, 11.354)] and caesarian section delivery [AOR = 2.826; 95% CI (1.618, 4.9360)] were found significantly associated with culture confirmed neonatal sepsis. Conclusion: Blood culture positivity rate of neonatal sepsis was high. Klebseilla pneumoniae and S.aureus were the predominant pathogens responsible for neonatal sepsis. Ciprofloxacin can be used as a treatment of choice for both Gram negative and positive bacteria. Key words: Antimicrobial Susceptibility, Bacterial profile, Ethiopia, Neonatal sepsis