Aims: To isolate a biosurfactant-producing bacterial strain and to identify and characterize the chemical structure and properties of its biosurfactants. Methods and Results: The bacterium Rhodococcus wratislaviensis BN38, isolated from soil, was found to produce glycolipid biosurfactants when grown on 2%n-hexadecane. The glycolipids were isolated by chromatography on silica gel columns and their structures elucidated using a combination of multidimensional NMR and ESI-MS/MS techniques. The main product was identified as 2,3,4,2'-trehalose tetraester with molecular mass of 876 g mol(-1). It was also noted that the biosurfactant was produced under nitrogen-limiting conditions and could not be synthesized from water-soluble substrates. The purified product showed extremely high surface-active properties. Conclusions: The glycolipid biosurfactant produced by the alkanothrophic strain R. wratislaviensis BN38 was characterized to be 2,3,4,2'-trehalose tetraester which exhibited high surfactant activities. Significance and Impact of the Study: Strain BN38 of R. wratislaviensis is a potential candidate for use in bioremediation applications or in biosurfactant exploration.