Aims The major aims of this study are to determine the capability of sulphur oxidizing bacterium (SOB‐1) to desulphurize dibenzothiophene (DBT) and crude oil, detection of the reaction kinetics and identify the proposed pathway of DBT desulphurization. Methods and Results The isolate was genetically identified based on 16S rRNA gene sequencing as Klebsiella oxytoca and deposited in the Genebank database under the accession number: MT355440. The HPLC analysis of the remaining DBT concentration revealed that, SOB‐1 could desulphurize 90% of DBT (0·25 mmol l−1) within 96 h. The maximum production of sulphate ions from the desulphurization of DBT (0·36 mmol l−1) and crude oil (0·4 mmol l−1) could be quantitatively detected after 48 h of incubation at 30°C. The high values of correlation coefficient (R2) obtained at all studied concentrations; suggested that biodesulfurization kinetics of DBT follows the first‐order reaction model. The kinetics studies showed that, DBT may have an inhibitory effect on SOB‐1 when the initial concentration exceeded 0·75 mmol l−1. The GC‐MS analysis exhibited four main metabolites rather than DBT. The most important ones are 2‐hydroxybiphenyl (2‐HBP) and methoxybiphenyl n(2‐MBP). Conclusions Klebsiella oxytoca SOB‐1 catalyzes the desulphurization of DBT through 4S pathway and forms four main metabolic products. The release of sulphate ion and formation of 2‐HBP indicating the elimination of sulphur group without altering the carbon skeleton of DBT. The bacterial strain could also catalyzes desulphurization of crude oil. The desulphurization kinetics follows the first‐order reaction model. Significance and Impact of the Study Klebsiella oxytoca SOB‐1 could be used as a promising industrial and environmental biodesulfurizing agent as it is not affecting carbon skeleton of thiophenic compounds and forming less toxic metabolic product (2‐MBP).