Biodesulfurization (BDS) has emerged as a sustainable and eco-friendly alternative to conventional sulfur removal methods for fossil fuels. This process utilizes microorganisms that leverage specific metabolic pathways, such as the 4S and Kodama pathways, to selectively break down sulfur-containing compounds like dibenzothiophene (DBT) while maintaining the integrity of the hydrocarbon structure. The 4S pathway involves a series of enzymatic reactions mediated by DszA, DszB, and DszC, converting DBT into non-toxic products. In contrast, the Kodama pathway employs different enzymes, resulting in the complete mineralization of sulfur. Although BDS offers significant advantages, its industrial application is limited by challenges such as slow reaction rates, substrate specificity, and the presence of inhibitory substances. Recent advancements in biotechnology, particularly genetic and metabolic engineering, are focused on improving the efficiency and resilience of desulfurizing microorganisms and their enzymes. Additionally, the use of omics technologies has provided valuable insights into microbial metabolic pathways and regulatory mechanisms, enabling the development of customized strains with enhanced desulfurization performance. Exploring alternatives like non-cellular biocatalysts and multi-enzyme systems also presents promising strategies to boost the efficiency of BDS processes. This chapter highlights the current state of research in biodesulfurization mechanisms and emphasizes the importance of overcoming existing challenges to advance the application of BDS technologies. As industries increasingly seek sustainable solutions to meet stringent environmental regulations, the continued exploration of innovative biotechnological approaches will be crucial for developing efficient methods for sulfur removal, ultimately contributing to cleaner fuel production and reducing the environmental impact of fossil fuel consumption.