Background: Conventional methods for isolation and antimicrobial susceptibility testing of bacterial isolates from positive blood culture bottles takes at least 48 hours. Direct antimicrobial susceptibility testing (AST) from positively flagged blood culture bottles helps to reduce the turnaround time (TAT) by 24 hours which will be useful in early initiation of appropriate antimicrobial therapy to reduce morbidity and mortality. Aims and Objectives: To evaluate the performance of direct AST from positively flagged BACTEC blood culture bottles and its comparison to conventional AST. Methods: A total of 356 blood culture bottles that were positively flagged on BACTEC 9120 were processed. Direct AST was performed from positive blood culture bottles. Bacterial isolates were identified using standard microbiological methods and tested against a wide spectrum of antimicrobial agents using the Kirby Bauer’s disc diffusion method following the Clinical & Laboratory Standards Institute (CLSI) guidelines. Direct and conventional AST results were compared and Categorical agreement (CA) with various errors was evaluated. Results: On direct Gram staining, 107 samples showed Gram negative bacilli and 105 showed Gram positive cocci and 144 samples showed growth of contaminants. A total of 1486 organism-antibiotic combinations were evaluated, out of which 1438 (96.76%) combinations showed CA which was extremely satisfactory. The categorical disagreement was found only in 48 (3.23%) of organism-antibiotic combinations; out of which major error was 23 (1.54%) followed by minor error 17 (1.14%) and very major error 8 (0.53%). Conclusions: The present study showed good concordance between the two methods and suggests the use of direct AST to reduce TAT by 24 hours for early initiation of therapy in patients with blood stream infections.