A biosurfactant-producing strain was isolated from the production water of an oil-field and was identified as Bacillus subtilis HOB2 by 16S rRNA gene sequencing. The production of biosurfactant by Bacillus subtilis HOB2 has been investigated using different carbon and nitrogen sources, under thermophilic and mesophilic conditions. The strain was able to grow and to produce surfactant, reducing the surface tension of medium to 27 mN/m on sucrose, and 28 mN/m on glucose after 24 h of cultivation. The strain was able to produce the maximum amount of biosurfactant when ammonium ions were used as nitrogen source. The surface-active compound was stable during exposure to elevate temperature (100°C), high salinity (25% NaCl) and a wide range of pH values (5.0-11.0). The biosurfactant was capable of forming a promising emulsification index (E24= 68%) with kerosene. The kinetic studies revealed that biosurfactant production is a cell growth-associated process. Preliminary chemical characterization revealed that the surfactant has a lipopeptide composition similar to surfactin as confirmed by TLC and IR analysis. Properties and characteristics of the biosurfactant produced by Bacillus subtilis HOB2 suggesting potential commercial applications, such as enhanced oil recovery, bioremediation of soil and marine environments, and food industries.