The biosurfactants are used by several industrial sectors such as petroleum, agriculture, food production, chemistry, cosmetics, and pharmaceuticals. Because of their hydrophobic and hydrophilic moieties, they have potency to reduce surface tension, interfacial tension between water-hydrocarbon systems, and low micelle concentration. Their characteristics strongly depend on the producer strain as well as on the medium composition, such as carbon and nitrogen sources. This study was conducted to investigate the influence of different sources of carbon (n-hexadecane, glycerol and glucose) and nitrogen (urea, NH₄Cl and NaNO₃) for the production of biosurfactants by a new strain of Pseudomonas aeruginosa B031 isolated from a rhizosphere of Paraserianthes falcataria L. Nielsen, a hardwood plant species at a phytoremediation field. The biosurfactant characteristics of the strain were evaluated, particularly its surface-active properties and potential to remove hydrocarbon. Glycerol was found to be the optimum carbon source, with rhamnose concentration, emulsification index, and critical micelle concentration (CMC) of 718 mg/L, 37%, and 35 mN/m, respectively. NaNO₃ was observed as the optimum nitrogen source, with rhamnose concentration, emulsification index, and CMC of 290 mg/L, 30%, and 24 mN/m, respectively. These biosurfactants efficiently reduced surface tension of culture broth from 42 mN/m to 31 mN/m for the glycerol treatment and from 37 mN/m to 24 mN/m for the sodium nitrate treatment. The crude biosurfactants from the glycerol and sodium nitrate treatments also removed 87.5% and 84%, respectively, of crude oil from sand. These rates were higher than those of the chemical surfactants (SDS and Triton X-100). These findings indicate that the biosurfactants produced by the strain from both glycerol and NaNO₃ treatments can efficiently decrease the interfacial tension of culture broth dilution and have a high emulsion index, thus hold promise in hydrocarbon bioremediation application.