This study reports characterization of a biosurfactant-producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS-8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant-producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum bio-surfactant production using 9% (v/v) inoculum at 30 degrees C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m(-1)) with a critical micelle concentration of >= 1.2 mg mL(-1). During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L-1 pure biosurfactant having significant emulsifying index (E-24, 70%) and oil-displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS-8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. (C) 2014 American Institute of Chemical Engineers