Background: Diagnosis of bloodstream infections in newborns is difficult due to a wide range of symptoms. Empirical therapy guided by a knowledge of the causative agents and their local antibiotic susceptibility profile is a crucial step in improving therapeutic results. As a result, we planned to investigate the bacteriological profile and antibiotic susceptibility pattern in neonatal sepsis. This was to compare the efficacy of a combination of ampicillin and gentamicin against 3rd generation cephalosporins for empirical antibiotic treatment of neonatal sepsis. Methods: It was a cross sectional observational study conducted over one and a half years.The period of study was from January 2019 to June 2020 in the Department of Paediatrics and Microbiology of a tertiary care Teaching hospital, Hydrabad. Blood culture samples of neonates suspected of having EOS or LOS were sent to the Microbiology department where they were inoculated into BACTEC TM Peds plus/F which was then inserted into the BD BACTEC fluorescent series instrument for incubation. Antibiotic sensitivity testing was done by disc diffusion as per CLSI guidelines. Zone sizes were measured and interpreted by BD PHOENIX AUTOMATED AST machine according to CLSI standards 2016. Results: 402 neonates were admitted to the neonatal unit of our hospital with suspected sepsis between January 2021 to June 2021. Out of which parents of 372 neonates consented to take part in the study. Out of which 196 were male and 176 were female neonates. Bacteria were isolated from 195 samples and 177 samples were negative out of listed 72 neonates. These 195 neonates were enrolled as cases in the study. Out of 195 cases, 75 cases were inborn and 120 were outborn. The blood culture isolation rate was 33.2 % and 56.5% in inborn and outborn respectively. There were 105 males and 90 females in the study. The culture positivity rate was 52.4%. Bacteria were isolated from 41 samples of suspected EOS neonates with a positivity rate of 33.8% and 154 samples of suspected LOS with a positivity rate of 34.5%. Gram-positive bacteria were isolated from the 107 cases and gram-negative bacteria were grown in 88 cases. The most common isolate was Staphylococcus aureus in 59(30.26%) followed by non- fermenters in 45(23.08%) cases. Conclusion: The prevalent pattern of causative etiological agents and their sensitivity pattern is critical because it aids in the selection of particular and effective antibiotic(s) for the index case's therapy. It also aids in the development of an institutional strategy for the selection of antibiotics for newborns admitted with suspected sepsis at the time of admission. This helps to avoid antibiotic abuse and the development of antibiotic resistance.