INTRODUCTION: India accounts for 30% of neonatal deaths globally. Bacterial sepsis is a major cause of morbidity and mortality in newborns. Prompt detection of microorganisms and early institution of therapy are of paramount importance. MATERIALS AND METHODS: The study was conducted in the Department of Microbiology over a period of one year. Two samples of blood were collected under aseptic precautions, 1 ml each was added to conventional blood culture bottle with biphasic media and paediatric BacT/ALERT bottle. Microorganisms were identified by Gram staining, standard biochemical tests and appropriate antibiograms. The common microorganisms responsible for early- and late-onset neonatal sepsis were identified, and the resistant strains were studied in detail. The main clinical presentations and maternal and neonatal risk factors associated with neonatal sepsis were identified and statistically correlated. RESULTS: Of the 233 newborns, 44 (18.9%) were culture positive, with higher incidence of sepsis in low birthweight male babies. Of the 44 isolates, 31 (70.5%) were Gram-negative organisms, with Klebsiella pneumoniae subspecies pneumoniae (45.5%) being the most common isolate. The prevalence of extended-spectrum beta-lactamase production in this study was 54.8%. Screening for AmpC production showed that 25.8% of the isolates were positive. The maternal risk factor of premature rupture of membrane of more than 18 h was seen in the case of 74 babies (31.8%), and 138 (59.2%) babies had prematurity as neonatal risk factor leading to sepsis. CONCLUSION: As the aetiological agents in neonatal sepsis vary in different circumstances and antimicrobial resistance due to different mechanisms is prevalent, antibiotic usage should be based on culture and sensitivity results.