Corn steep liquor (CSL) is a liquid byproduct generated by the corn wet-milling industry. This agroindustrial residue is obtained when corn is digested with warm water and SO2 in the presence of different spontaneously growing microorganisms. The microbial population of CSL includes lactic acid bacteria, which are widely known to produce biosurfactants. However, there is no information available about the possible existence of surface-active compounds in CSL. In this work the surface tension activity of CSL was evaluated and the biosurfactant contained in this residue was extracted with different organic solvents and characterized. The biosurfactant obtained from CSL was analyzed by Fourier transform infrared spectroscopy, and the composition in fatty acid was analyzed by gas chromatography-mass spectrometry. The hydrophobic chain of the biosurfactant contained in CSL comprised 50-55.2% linolelaidic acid, 15.7-22.2% oleic and/or elaidic acid, 5.9-14.6% stearic acid, and 14.9-19.6% palmitic acid.