A soil isolate, identified as Burkholderia gladioli OR1, has been reported in our earlier study, to possess broad spectrum antimicrobial activity against drug resistant clinical isolates of bacteria and fungi. The 16S rRNA gene sequence data of B. gladioli strain OR1 has been deposited in the GenBank databases (MTCC) under the accession number MTCC11251. Thin layer chromatography and bioautography studies of chloroform extract of the cell free supernatant of this organism, showed at least four different antimicrobial compounds. The silica gel column chromatography and high performance liquid chromatography led to the isolation of one compound that exhibited growth inhibitory activity against multi-antibiotic resistant clinical isolates of Staphylococcus aureus (resistant to amikacin, ciprofloxacin, clindamycin, clinafloxacin, erythromycin, gentamicin and methicillin). On the basis of mass, infra-red (IR), 1H nuclear magnetic resonance (NMR), 13C NMR and 2D NMR spectral studies, we propose the structure of this antibiotic to be bis-(2-ethylhexyl) phthalate, isolated for the first time from the species of the genus Burkholderia.