Multidrug resistant (MDR) gram negative bacterial infections are most prevalent in the hospitals which are truly untreatable and remedial verdict are very deprived. In the current investigation, two compounds (Quercetin and Pinitol) and fractions have been isolated and characterized from Indigofera barberi using chromatographic and NMR studies. Antimicrobial assays reveal IB-Me, IB-D and IB-E fractions and quercetin have shown eloquent antimicrobial activity on clinically isolated bacteria such as Pseudomonas aeruginosa, E. coIi, Kiebsiella pneumonia and Citrobacter sp. Molecular docking studies demonstrated quercetin has exhibited highest binding affinity with DNA gyrase B and subsequently eleven pharmacophore features viz. five HBD/HBA, two hydrophobic, one HBA and three aromatic (Pi) rings were identified using MOE. Pharmacophore based virtual screening, docking, binding energies and binding affinity with Born-volume (GB/VI) studies demonstrated thirteen lead molecules which were specifically interacted with Asn46, Asp73, Arg76, Thr163, Lys103, Arg136 residues and conserved water molecules within ATP pocket of DNA gyrase B. These scaffolds comply with Lipinski rule of five, toxicity and drug likeness properties deliberated as drug compounds. Hence, these compounds could be endorsed as persuasive DNA gyrase B inhibitors which can be considered as novel compounds in the clinical management of MDR Gram negative bacterial infections. (C) 2016 Elsevier Ltd. All rights reserved.