AIMS: In this study, an attempt has been made to isolate and identify the bioactive compounds from hydroalcoholic extract of Terminalia chebula fruits effective against multidrug-resistant uropathogens and also to elucidate the influence of metal ions on the growth inhibitory activity of isolated compounds against the studied bacteria, if any. METHODS AND RESULTS: Bioassay-guided fractionation and extensive spectrometric analyses (FT-IR, (1) H NMR, (13) C NMR and ESI-MS) were used to isolate and characterize the bioactive compound. Growth inhibitory activities of isolated compound were studied by agar well diffusion and microbroth dilution assay methods. Checkerboard titration method was used for combination study between antibiotics and isolated compound. Influence of metal ions on growth inhibitory activity of this bioactive compound against the test isolates were also studied by INT [P-iodonitrotetrazolium violet; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride] colorimetric assay. The isolated bioactive compound 1, 2, 6-tri-O-galloyl-?-D-glucopyranose was found to be responsible for antibacterial activity against multidrug-resistant uropathogens and showed synergy with trimethoprim and gentamicin. This antibacterial activity of bioactive compound was counteracted by the supplementation of iron in the medium. CONCLUSION: Terminalia chebula fruit extract contains bioactive compound effective against multidrug-resistant uropathogens, and this antibacterial activity may be due to its iron-complexing property. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, the antibacterial activity exhibited by isolated gallotannin against multidrug-resistant uropathogens is first time reported by us. Besides, these promising findings may lead to the development of antimicrobial agents from T. chebula fruits for the treatment of urinary tract infections caused by these pathogens.