In the present study, characterization and antibacterial activity of a glycoside antibiotic from fermentation broth of bioactive Streptomyces variabilis PO-178 recovered previously from Western Ghat soil of Agumbe, Karnataka, India was investigated. Mass cultivation of the strain PO-178 was carried out in Starch casein nitrate broth. The fermentation broth was extracted using butanol solvent in a separation funnel. The concentrated butanol extract was subjected to chromatographic techniques viz., thin layer and silica gel column chromatography for purification of bioactive components. Out of 3 fractions (A, B and C) obtained, only one fraction (C) displayed inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa. The bioactive fraction C was subjected to spectral analysis (LC-MS, IR, 1 H-NMR and 13 C-NMR) in order to determine the structure. On the basis result of spectral analysis, the purified fraction was predicted as a glycoside antibiotic with molecular weight 514 and molecular formula C 24 H 34 O 12 . The antibiotic displayed marked inhibitory activity against S. aureus as revealed by wider zones of inhibition and low minimum inhibitory concentration value.