The fermentative production of rhamnolipid biosurfactant from Pseudomonas aeruginosa MTCC 2297 was carried out by submerged fermentation using various cost-effective waste materials such as orange peelings, carrot peel waste, lime peelings, coconut oil cake, and banana waste. The orange peel was found to be the best substrate generating 9.18 g/l of rhamnolipid biosurfactant with a surface tension reduction up to 31.3 mN/m. The production was growth independent, and optimum conditions were standardized. The emulsifying activity was highest against kerosene (73.3%). Rhamnolipid components were purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, high-performance liquid chromatography and thin-layer chromatography. The major rhamnolipid components were characterized, by fast atom bombardment mass spectrometry, as a mixture of dirhamnolipids and monorhamnolipids.