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Introduction: Bacterial Sepsis by Multidrug Resistant Gram Negative Bacilli (MDRGNB) producing Extended Spectrum β-Lactamases (ESBL) is one of the major causes of mortality and morbidity in hospitals. Early detection of ESBLs directly from positive blood cultures can reduce mortality. The phenotypic detection of ESBLs is difficult as they may be masked by the co-production of additional enzymes like AmpC. This can be overcome by using an Aztreonam Discs With and Without Clavulanate (AO/CL) method. Aim: To identify ESBLs directly from the positive blood cultures by using AO/CL disc diffusion method and to detect the genes coding for ESBL enzymes by conventional Polymerase Chain Reaction (PCR). Materials and Methods: A prospective study was conducted over a period of five months (October 2020-February 2021). A total of 100 positive blood cultures showing Gram negative bacilli on Gram stain was subjected to direct detection of ESBLs by using Cefotaxime (CTX), Ceftazidime (CAZ) discs with and without clavulanate and AO/CL. Isolates from positive blood culture were identified to genus and species level by VITEK-2 compact. Isolates were tested for ESBL production by CAZ/ CTX with and without clavulanate disc diffusion method as recommended by CLSI. PCR was carried out to detect target genes responsible for ESBL production such as CTX–M, TEM, SHV genes. Statistical analysis was done by using MS Excel sheet. Descriptive statistics like percentage calculation was done in the study. Results: Out of 100 positive blood cultures showing Gram Negative Bacteria (GNB) on Gram stain, 33 were positive for ESBL production by direct disc diffusion method. Out of these, 27 ESBL producers were detected by CAZ/CTX with and without clavulanate disc diffusion method and AO/CL method whereas 6 ESBL producers were detected by AO/ CL disc diffusion method only. A 27 culture isolates were found positive for ESBL production by CAZ/CTX with and without clavulanate disc diffusion method as recommended by Clinical and Laboratory Standards Institute (CLSI). Out of 33, 28 (85%) isolates possessed one of the target genes for ESBL production such as 10TEM (36%), 10CTX-M (36%), 07TEM+CTX M (25%), 01SHV (3%). Conclusion: Direct detection of ESBLs plays a significant role in management of sepsis. It helps the clinician in escalation and de-escalation of antibiotics and prevents the development of antimicrobial resistance. It contributes towards antibiotic stewardship and better compliance to infection prevention and control protocols. AO/CL method is preferred to detect ESBL producers directly from positive blood culture bottles.