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The novel isolate Pseudozyma sp. NII 08165 produced glycolipid biosurfactants, which was a combination of all the three mannosylerythritol lipids (MELs) isomers along with some unknown glycolipids. The strain produced 34 g/l MELs in medium containing 8% (w/v) soybean oil as carbon source after nine days of fermentation. The structural characterization of purified MEL revealed the hydrophobic structure of MEL-C consisting of short chain fatty acid (C2 or C4) at the C-2′ position and a long chain fatty acid (C14, C16 or C18) at the C-3′ position of the mannose moiety. The MEL-C showed good surface activity with critical micelle concentration (CMC) of 4.5 × 10⁻⁶ M and surface tension of 33 mN/m at CMC. The crude biosurfactants were stable at high temperature and over the alkaline pH range which favour their scope of application as laundry detergent additives. Fabric wash analysis revealed that crude …