Background: This study's objectives are to identify and determine the prevalence of ESBLs producing gram-negative isolates from cases of neonatal sepsis that occurred in Indore, Madhya Pradesh; and also, to investigate the detection of antibiotic susceptibility pattern of gram-negative bacilli isolated in blood culture of neonatal sepsis. Methods: In terms of its methodology, the research can be categorized as an experiment that also consists of a descriptive component. Both of these components are included in the investigation. After receiving approval on an ethical level from the Institutional Ethical Committee of the Index Medical College Hospital & Research Centre (IMCH&RC), which can be found in the sentence before this one, it was carried. Inclusion criteria: Neonates under 60 days old with a clinical history of sepsis were admitted to the Index Medical College Hospital & Research Centre in Indore for blood cultures. Neonatal blood was drawn. The blood was cultured for further examination. The study included inborn and out born infants who were admitted to the IMCH and RC's Neonatal Intensive Care Unit (NICU) with suspected clinical features of sepsis at admission or developed such features after admission for other reasons. The study included infants admitted to the NICU and other IMCH and RC departments. The infants were given antibiotics even though it was unclear. Results: 50% of the GNB isolates were positive for ESBL. ESBL positivity was significantly more prevalent (>50%) in E. coli, Klebsiella spp., and Enterobacter spp. than in Acinetobacter spp. (1%). Similarly, 89% of E. coli, Klebsiella spp., and Enterobacter spp.were MDR, with Acinetobacter spp. There was a variation in susceptibility status according to specific bacterial genus and antimicrobial agent, but in general, fifty five percent of the GNB isolates that were tested showed resistance to the antimicrobials that were included in the first-line AST panel. Conclusion: The present study concludes that MDR bacteria, ESBL, and carbapenemase-encoding AMR genes in GNB make patient treatment difficult