Aims: To isolate a biosurfactant-producing bacterium and find new products within its culture. Methods and Results: A biosurfactant-producing bacterium identified as Rhodococcus erythropolis (3C-9 strain) was isolated from seaside soil. When n-hexadecane was supplied as the sole carbon source, two types of biosurfactants (free fatty acids and glycolipids) were detected in the supernatant of the bacterial culture by use of thin layer chromatography (TLC). Gas chromatographymass spectrometry (GC-MS) analysis revealed that the former consisted of at least 12 free fatty acids of chain lengths from C9 to C22; and the latter contained 2 kinds of glycolipids (a glucolipid and a trehalose lipid), which were detected by use of TLC, as well as GC-MS. The hydrophobic moieties of both glycolipids consisted of seven types of straight-chain fatty acids of varying compositions, with chain lengths ranging from C-10 to C-18-It was also noted that biosurfactants of strain 3C-9 were produced in a manner that was growth-related and cannot be synthesized from water-soluble substrates. The effects to enhance the solubility of polycyclic aromatic hydrocarbons and the degradation rate of hexadecane were also tested. Conclusions: The biosurfactants produced by strain 3C-9 of R. erythropolis included two kinds of glycolipids, as well as free fatty acids. These biosurfactants were notably different from those of previously reported Rhodococcus species, both in terms of their structure and chemical composition. Significance and Impact of the Study: Strain 3C-9 of R. erythropolis is a competitive candidate for use in oil spill cleanup operations, or in new biosurfactant exploration. The findings in this report show that Rhodococcus is a natural reservoir of new biosurfactants.