Microorganisms capable of producing biosurfactants can be isolated by a series of steps including hydrocarbon enrichment culture, hemolytic activity assay on blood agar plates and oil displacement activity assay etc. Ten strains(including bacteria, yeasts and fungi) were isolated with higher surface activity from 1000 samples from soil, sludge, waste water etc. Two strains of bacteria produced rhamnolipids and trehaloselipids respectively. One strain of bacterium produced long chain unsaturated carboxylic acid and another produced azelaic acid. Two strains of yeasts produced lipopolysaccharides with excellent emulsification property.