Microorganisms can metabolize or transform a range of known chemical compounds present in fossil fuels by naturally having highly specific metabolic activities. In this context, the microbial desulfurization of fuels is an attractive and alternative process to the conventional hydrodesulfurization (HDS) process, since the thiophenic sulfur containing compounds such as dibenzothiophene (DBT) and benzothiophene (BT) cannot be removed by HDS. A DBT desulfurizing mesophilic bacterium, identified on the basis of 16S rRNA gene sequence as Gordonia sp. HS126-4N (source: periphery soil of a coal heap) has been evaluated for its biodesulfurization traits and potential to desulfurize the thiophenic compounds. The HPLC and LC/MS analyses of the metabolites produced from DBT desulfurization and PCR-based nucleotide sequence confirmation of the key desulfurizing genes (dszA/dszB/dszC) proved that HS126-4N could convert DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The isolate could convert 0.2 mM of DBT to 2-HBP within 48 h and was reasonably tolerant against the inhibitory effect of 2-HBP (retained 70% of growth at 0.5 mM 2-HBP). The isolated biocatalyst desulfurized/degraded 100% of 0.2 mM of 4-methyl DBT, 2,8-dimethyl DBT, BT and 3-methyl BT within 108 h. The capabilities to survive and desulfurize a broad range of thiophenic sulfur containing substrates as well as less inhibition by the 2-HBP suggest that HS126-4N could be a potential candidate for improved biodesulfurization/organic sulfur removal from fossil fuels.