Surfactants play a very important role in laundry and household cleaning products ingredients. In this research, the application of lipopeptide biosurfactants, produced by Bacillus subtilis SPB1, in the formulation of a washing powder was investigated. The SPB1 biosurfactant was mixed with sodium tripolyphosphate as a builder and sodium sulfate as filler. The efficiency of the formulated detergent composition with different washing conditions to remove a stain from cotton fabric was examined. The results showed that the formulated detergent was effective in oil removal, with optimal washing conditions of pH, temperature, striate and time of washing system of 7, 65°C, 1000 RPM and 60 min, respectively. A comparative study of different detergent compositions (biosurfactant-based detergent, combined biosurfactantcommercial detergent, and a commercial detergent) for the removal of oil and tea stains, proved that the bio-scouring was more effective (>75%) in terms of the stain removal than the commercial powders (<60%). Moreover, the results demonstrated that the biosurfactant acts additively with a commercial detergent and enhances their performance from 33 to 45% in removing oil stain and from 57 to 64% in removing tea stain. As a conclusion, in addition to the low toxicity and the high biodegradability of the microbial biosurfactants, the results of this study have shown that the future use of this lipopeptide biosurfactant as laundry detergent additive is highly promising.