Glycolipids produced byPseudomonas aeruginosa YPJ-80 were characterized by chromatographic and spectroscopic techniques as a mixture of two rhamnolipids. For recovery of glycolipids from the culture broth, various isolation methods including ultrafiltration, adsorption and solvent extraction were compared. Ultrafiltration showed the best results in terms of glycolipids recovery. Further purification for spectroscopic analysis was carried out by adsorption chromatography and preparation thin layer chromatography. From the spectroscopic analysis, such as IR spectroscopy, FAB-MS,1H-NMR and13C-NMR and hydrolysis analysis, the glycolipids were identified as L-α-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate and 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate. Monorhamnolipid and dirhamnolipid lowered the surface tension of water to 28.1 mN/m and 29.3 mN/m, respectively.