Introduction: Neonatal sepsis is one of the leading causes of mortality and significant morbidity among neonates globally. Formulating Neonatal Intensive Care Unit (NICU) antibiotic policy on a timely basis is the need of hour. This study is aimed at identifying the primary pathogens associated with neonatal sepsis and establishing antibiotic policy based on bacteriological profile and their antibiotic sensitivity. Methods: This is a retrospective study done in a tertiary care referral hospital in South India. The study period was from Sept'17 to Feb'18. The Inclusion criteria were to include all blood culture positive samples of neonates admitted in NICU. Results: There were totally 264 positive cultures during the study period. The gram negative flora predominates with 69.7%. Of the gram negative growth klebsiella was the most common pathogen and proteus was the least common pathogen. There was 30.3% of gram positive growth with CONS as predominant pathogen. Other gram positive bacteria were Enterococcus and staphylococcus. On analysis of the antibiotic sensitivity pattern, more than 50% of klebsiella growths were sensitive to amikacin, ciprofloxacin, cefoperazone and piperacillin. The gram negative isolates were resistant to ampicillin, ceftriaxone, cefotaxime and imipenem. The gram positive isolates were more sensitive to ciprofloxacin, linezolid, piperacillin and cefoperazone. The gram positive isolates of CONS and staphylococcus were less sensitive to vancomycin. Conclusion:The present study reinforces the wide diversity of bacterial isolates in neonatal sepsis and reiterates the need for periodic surveillance of microbial flora. The growing resistance to the antibiotics like vancomycin is of serious concern.