Fusarium dry rot is considered to be the most critical soilborne and postharvest disease that damages potato tubers worldwide when they are stored for a long time. This study was performed to demonstrate the effect of crude extract, culture filtrate, and cell suspension obtained from the bacterium Bacillus subtilis Hussain T-AMU, on the net house and field. From oil-contaminated soil, through the serial dilution method, biosurfactant bacteria were isolated on nutrient agar medium. To isolate and screen the prospective biosurfactant strain, various biosurfactant screening methods were used. Standard protocols were carried out for morphological, molecular, and chemical characterization. The disease incidences were significantly decreased under net house and field conditions after treatment with biosurfactant extract (20%, 70%), culture filtrate (23%, 49%), and bacterial cell suspension (46%, 27%), respectively. These results indicate a significant effect of B. subtilis HussainT-AMU against Fusarium oxysporum. The characterization of the partially purified extract by gas-chromatography indicated the existence of a high amount of methyl ester fatty acids. The quantitative analysis determined the presence of lipopeptide-surfactin, fengycin, and iturin in the biosurfactant extract. Scanning electron microscopy revealed that morphological variation was revealed in the structure of stomata size in leaves and in the mycelia of the pathogen that was investigated. This promising biocontrol agent can be a plausible option for further research and can also be used for the production of green biomolecules at an additional industrial level.