Background: Bridelia scandens Wild. (Euphorbiaceae) leaves are widely used to cure asthma, bronchitis pleurisy, exudation, sores in mouth and genital cancers. Objective: To evaluate antibacterial activity of the leaf calli methanol extract (LCME). Materials and Methods: Mass production of leaf calli was established on MS medium supplemented with 0.5 mg/L BAP and 0.5 mg/L 2, 4-D. Methanol extract of the dried calli was subjected to HR-LCMS analysis, antibacterial screening of the extract was carried out against human pathogenic clinical isolates. Molecular docking study of HR-LCMS identified compounds was performed by docking with bacterial enzyme DNA gyrase. Results: HR-LCMS analysis of LCME shows that the compounds azaperone bifonazole, fusidic acid, lasalocid and quinine as the major constituents. The antibacterial screening of LCME against clinical pathogens showed significant bactericidal activity against the strains Staphylococcus aureus (17.67±0.88 mm.d.), Streptococcus pneumonia (13.67±0.33), Pseudomonas aeruginosa (16.33±0.67), Salmonella typhi (17.67±0.33), and Vibrio cholera (15.33±0.33) as compared to the standard drug ciprofloxacin. The molecular docking of lasalocid against the bacterial enzyme DNA gyrase exhibited good binding affinity of -4.9 kcal/mol, good drug likeness (2.5589), 2 hydrogen bonds and hydrophobic interaction with 7 amino acid residues, so that lasalocid processes good inhibitor as compared to other 4 compounds. Conclusion: LCME of Bridelia scandens showed significant antibacterial activity against Staphylococcus aureus and Salmonella typhi. Lasalocid is the major phytocomponent of LCME which exhibited good inhibitory activity against bacterial enzyme DNA gyrase. This investigation supported traditional claim of LCME as potential antibacterial drug.