Abstract: Ten thermophillic bacterial isolates were screened for their ability to produce biosurfactant depending on the emulsification activity, emulsification index and surface tension. The results showed that Geobacillus thermoleovorans (JQ 912239) was the most efficient one for biosurfactant production. The chemical composition of partial purified biosurfactant revealed that it consists of 37.7% lipids, 26.2% carbohydrate and 10.7% protein. The partial and/or purified biosurfactant was subjected to Fourier Transform Infrared Spectroscopy (FTIR), High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and Nuclear Magnetic Resonance (NMR) to complete the chemical characterization. The results of the FTIR confirmed that the biosurfactant contains the lipid, carbohydrate and protein. The HPLC analysis revealed that the lipid fraction consists of palmitic acid, stearic acid and oleic acid, the carbohydrate part consists of xylose, mannose and maltose and the protein part consists of three amino acids (aspartic acid, glutamic acid and glutamine). The lipid fraction was further analyzed using GC and NMR. GC analysis indicated that it contains palmitic acid methyl ester (c16:0), stearic acid (c18:0) and oleic acid (c18:1n9c) however, purified biosurfactant contains a high percentage of palmitic acid methyl ester. The NMR analysis for purified biosurfactant detected that the main component of lipid fraction is triglycerides.