ABSTRACT: The present study aimed to isolate the high-efficiency petrol metabolizing thermophilic bacteria from petrol contaminated soil samples. Isolation was carried out through enrichment culture, serial dilution and pour plate methods using the petrol supplemented minimal salt media. The isolated bacteria were analyzed to document growth behavior, petrol removal efficiencies, antibiotic resistance profile, and biochemical characteristics. The 16S rRNA based phylogenetic analysis helped to reveal the identity of isolated bacterial species and construct the phylogenetic trees. Total nine bacteria were isolated, out of which three (IUBP2, IUBP3, IUBP5) were identified as Brevibacillus formosus, one (IUBP1) was found similar to Brevibacillus agri, four (IUBP7, IUBP8, IUBP13, and IUBP14) shared homology with Burkholderia lata, and one (IUBP15) with Burkholderia pyrrocinia. All the isolates were fast growing and exhibited considerable petrol degradation potential. The highest petrol removal efficiency (69.5% ± 13.44/6 days) was recorded for the strain IUBP15 at a petrol concentration of 0.1% (v/v). All bacteria studied (100%) were positive for esculinase and phosphatase. Many strains exhibited positive responses for arginine dehydrolase (22%), ?-naphthylamidase (11%), ?-D-glucosaminide (33%), mannitol (55%), sorbitol (66%) and inulin (88%) fermentation test. While all were sensitive to the antibiotics, some of them were found resistant against chloramphenicol and oxacillin. The remarkable biochemical characteristics and considerable petrol removal potential (40-70%) highlights utilization of the bacteria isolated for petrol bioremediation, mineralization of organophosphates, dairy and food industry, and also as biofertilizers and biocontrol agents.The present study aimed to isolate the high-efficiency petrol metabolizing thermophilic bacteria from petrol contaminated soil samples. Isolation was carried out through enrichment culture, serial dilution and pour plate methods using the petrol supplemented minimal salt media. The isolated bacteria were analyzed to document growth behavior, petrol removal efficiencies, antibiotic resistance profile, and biochemical characteristics. The 16S rRNA based phylogenetic analysis helped to reveal the identity of isolated bacterial species and construct the phylogenetic trees. Total nine bacteria were isolated, out of which three (IUBP2, IUBP3, IUBP5) were identified as Brevibacillus formosus, one (IUBP1) was found similar to Brevibacillus agri, four (IUBP7, IUBP8, IUBP13, and IUBP14) shared homology with Burkholderia lata, and one (IUBP15) with Burkholderia pyrrocinia. All the isolates were fast growing and exhibited considerable petrol degradation potential. The highest petrol removal efficiency (69.5% ± 13.44/6 days) was recorded for the strain IUBP15 at a petrol concentration of 0.1% (v/v). All bacteria studied (100%) were positive for esculinase and phosphatase. Many strains exhibited positive responses for arginine dehydrolase (22%), ?-naphthylamidase (11%), ?-D-glucosaminide (33%), mannitol (55%), sorbitol (66%) and inulin (88%) fermentation test. While all were sensitive to the antibiotics, some of them were found resistant against chloramphenicol and oxacillin. The remarkable biochemical characteristics and considerable petrol removal potential (40–70%) highlights utilization of the bacteria isolated for petrol bioremediation, mineralization of organophosphates, dairy and food industry, and also as biofertilizers and biocontrol agents.