Oil seeds sector is one of the major dynamic components of the agriculture world. Oil seeds such as canola (Brassica napus) require a higher quantity of sulfur (S), which is supplied through inorganic fertilizers. However, the overapplication of agro-chemicals to get higher yields of crops is harming the soil health. Therefore, the application of bacterial cultures with plant growth-promoting activity as biofertilizers ensures soil health maintenance and enhances crop productivity. To achieve this aim, the present research was initiated by procuring three sulfur-oxidizing bacteria (SOBs), namely, SOB 5, SOB 10, and SOB 38, from the Microbiology Department, PAU. In the initial assessment, all three SOB cultures showed resilience to pesticide toxicity at the recommended dosage, with the exception of ridomil. These cultures were later characterized morphologically, biochemically, and at the molecular level using 16s rRNA resulting in their identification as Enterobacter ludwigii strain Remi_9 (SOB 5), Enterobacter hormaechei strain AUH-ENM30 (SOB 10), and Bacillus sp. 5BM21Y12 (SOB 38). Functional characterization of these SOB cultures revealed their ability to exhibit multifarious plant growth-promoting traits. Bacillus sp. 5BM21Y12 showed greater functional activity, including high P solubilization (14.903 µg/mL), IAA production (44.28 µg/mL), siderophore production (13.89 µg/mL), sulfate ion production (0.127 mM), ammonia excretion (2.369 µg/mL), and Zn solubilization (22.62 mm). Based on the results of functional and molecular characterization, Bacillus sp. 5BM21Y12 was selected for field trials by formulating different treatments. Composite treatment, T8 (100% S + Bacillus sp. + pesticides) significantly enhanced growth parameters (plant height, root, and shoot biomass), yield attributes (siliqua length, test weight, number of siliqua/plant), yield parameter (total biomass and seed yield), quality parameter (crude protein and oil) as compared to all other sole treatments employed in the field. A combined application of non-pathogenic Bacillus sp. 5BM21Y12, with good functional activity enhanced yield of crop due to synergistic and additive interaction with fertilizer/pesticides. As biofertilizer application reduces the input of pesticides/fertilizers new inoculant formulations with cell protectors and the development of compatible pesticides should be searched to assure the benefits of integrated treatment.