This study aimed to produce and characterize biosurfactants using the Thermoanaerobacter sp. CM-CNRG TB177 strain isolated from an oil field in Mexico, as well as assessing the influence of different carbon and nitrogen sources on the capacity of the produced surfactant to reduce the surface tension of water. The thin-layer chromatography (TLC) revealed that the obtained extract corresponds to a mono-rhamnolipid; the results of the ultra-performance-liquid chromatography/mass spectrometry (UPLC/MS) analysis revealed that the Thermoanaerobacter sp. CM-CNRG TB177 strain produces a mixture of three rhamnolipids, whose masses correspond to mono- rhamnolipid. The rhamnolipids mixture obtained using 2.5% molasses as carbon source diminished the surface tension of water to 29.67 mNm(-1), indicating that the concentration of molasses influenced the capacity of the produced surfactant to reduce the surface tension of water. Also, the microorganism was not capable of growing in the absence of yeast extract as nitrogen source. To the best of our knowledge, the presented results describe for the first time the nature of the biosurfactant produced by a bacterium of the Thermoanaerobacter genus.