Biosurfactants, also known as biological surface-active agents, are a group of surface-active agents, which are produced by a variety of microorganisms. Biosurfactants possess the characteristic property of lowering surface tension, increase the solubility of poorly soluble compound, low toxicity, non-allergenic, and biodegradable. Growing public awareness about the environmentally friendly health care and associated product has stimulated the search for eco-friendly compounds in laundry detergents. In this research, the application of biosurfactants in the formulation of a washing detergent was investigated. Variation of biosurfactants and its mixture with sodium tripolyphosphate as a builder and sodium sulfate as filler was applied to wash cotton fabric which was contaminated with known amounts of stain (chocolate milk). The effects of various bio-detergent formulations, including the ratio of biosurfactants and builder, was examined. The formulation presented in this study was also compared with some standard detergent for the stains removal efficiency. The results showed that the rhamnolipid biosurfactants have a promising as a substitute for its synthetic counterpart. Based on the colorfastness to wash, color strength (K/S), and color difference (∆E) value, rhamnolipid based bio-detergent have similar washing effectiveness compared to the synthetic detergent. Research result also found that Na-bicarbonate and Na-citrate have the potential as a substitute for phosphate-based detergent builder.