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Introduction: Microorganism present in blood whether continuously or intermittently are threat to every organ in the body. The surveillance of etiological agents in these infections is essential for their prevention and treatment. Awareness of the baseline microbial resistance specific to a hospital prevents irrational use of antibiotics in that hospital. Thus helps progress a step forward in the prevention of spread of antibiotic resistance. Material and methods: A retrospective study was conducted in the department of Microbiology. During the study period, blood samples collected from all age group OPD, IPD and ICU patients suspected of bacteremia and septicemia were analyzed. All Gram-negative bacilli, Gram-positive cocci and Yeast were investigated while anaerobic bacteria and cultures with mixed growth were excluded. Results: During the study period of Jan 2017-Dec2017, 1885 blood cultures were analyzed. 305 (16.1%) were found to be positive, out of which 236 were from ICU, 58 were from IPD and 11 from OPD. Among Gram positive cocci, CoNS is commonest followed by Staphylococcus aureus whereas in Gram negative bacilli, Klebsiella pneumoniae was commonest organism followed by Pseudomonas aeruginosa. In our study, 90% of Enterobacteraceae were ESBL producers. MRSA were isolated in 50% and MRCoNS in 71%. Gram-positive isolate were least sensitive to penicillin (10%) while it was most sensitive to tigecycline in 100% followed by vancomycin in 95%. Gram negative isolates of Enterobacteriaceae were least sensitive to Aztreonam (11%) and while it was most sensitive to Colistin in 87% of cases. Candida species were isolated in 54 (18%) of which NICU accounted for majority of cases. Candida albicans was least sensitive to Fluconazole (82%) and Non albicans candida to Amphotericin B (70%) while they were sensitive to all other antifungals. Conclusion: The retrospective study conducted showed both gram positive and gram negative bacteria were responsible for blood stream infections. Most of the strains were multi drug resistant. Rapid isolation and identification of pathogens by automated blood culture system and antibiogram with minimum inhibitory concentration (MIC) value provides early and appropriate treatment to the seriously ill patients leading to reduce mortality and reduce duration of hospitals. Resistance flagging of the bacterial isolates guides us to perform barrier nursing and isolate the patient to prevent spread of infection. The daily analysis of resistance flagging and MIC values give important information for choosing selective antibiotics leading to good antibiotic stewardship which in turn reduces patient morbidity and mortality. How to cite this article: Manish Kumar Rout, BibhuduttaRautaraya. Bacteriological profile, antibiogram and phenotypic resistance flagging of blood culture isolates by automated methods in a tertiary care hospital.