Bloodstream infections (BSIs), particularly in neonates, must be diagnosed and treated with great care. Blood cultures continue to be the gold standard for diagnosis and are one of the most frequent causes of morbidity and mortality in BSIs. This study aims to estimate the bacteriological profile in blood culture among neonates in the tertiary care NICU of western Uttar Pradesh. This is a retrospective study, done in tertiary care hospital. All the neonatal blood samples, received during the period of 1st December 2020 to 30th November 2022 are included in the study. All samples were incubated in BacT/Alert system. The positive blood culture was further plated on Blood Agar & MacConkey Agar. The growths were identified according to the standard guidelines and the antibiotic sensitivity was performed by Kirby–Bauer’s disc diffusion method. A total of 637 neonatal blood samples were received during the period of 1st December 2020 to 30th November 2022. Among them, 15.2% of blood cultures were positive by BacT/Alert blood culture system of sepsis in neonates, Out of these total 52.6% patients had a Gram-negative bacterial infection, and 47.4% patients had Gram-positive bacterial infection. Among these 97 blood culture-positive cases, CoNS were isolated in 29.9%, followed by Klebsiella species in 20.6% cases, 18.5% Escherichia coli, 15.5%. Staphylococcus aureus,6.2% Pseudomonas spp., 5.2% Acinetobacter spp, 2.1% Enterococcus spp and 1% Enterobacter & Citrobacter each respectively. Our study shows 15.2 % of positive blood cultures. Klebsiella species and E. coli were found to be more prevalent among gram-negative bacteria. Among gram-positivemost common organisms were CoNS and Staphylococcus aureus. So it is important to include good hygiene practices and standard protocol in paediatrics wards to minimize the chances of bacterial sepsis.