The present study was held to optimize production, purify and identify biosurfactant from yeast strain Geotrichum candidum MK880487. Biosurfactant production was done on modified Hua medium and optimized in shake-flask method. Soybean oil (8%, v/v), KNO3 (0.75 g L−1) and yeast extract (0.3 g L−1) were the best carbon and nitrogen sources, respectively, with C:N ratio of 200:1, pH 8 at 30 °C for 168 h. Production scale-up was achieved on 2.5 L bioreactor as batch method. The resulting crude biosurfactant extract with yield of 1.75 g L−1 was purified by reversed phase column preparative HPLC. The resulting most active fraction F47 significantly reduced water surface tension by 51.92%. Chemical characterization using TLC, GC-MS, FTIR and LC-MS/MS methods revealed the biosurfactant to contain mainly glycolipid structure existing as mixture of Icariside F2, Cardenolide Di-Hexopyranoside and Di Galactosyl Di Acyl Glycerol glycolipid that collectively showed potential antifungal activity toward Macrophomina phaseolina.