A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography-mass spectrometry (GC-MS) to be a mixture of L-ornithine lipids, which were composed of L-ornithine and a different couple of iso-3-hydroxyfatty acid (C-15-C-17) and iso-fatty acid (C-15 or C-16) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.