The oil industries are mainly responsible for the production of oily waters due to the drilling and extraction of petroleum, generating major environmental disturbances. The use of flotation as a separation process of oily waters has been described, although it has been sometimes criticized due to the toxicity of collectors. The development and use of biodegradable surfactants may enhance the further acceptance of this separation technology. Biosurfactants, molecules produced by microorganisms present as a sustainable and promising technology in increasing flotation efficiency by being biodegradable and non-toxic. It is also observed among the biosurfactants characteristics that promote the destabilization or stabilization of emulsions of the water-oil type, the first being the reason why some species can be used as auxiliaries of the flotation process. Thus, this study investigated the potential application of a biosurfactant in the separation of residual oil using a prototype of DAF. The biosurfactant was produced by Candida guilliermondii UCP 0992 grown in a low-cost medium and formulated with 4.0% of corn steep, 2.5% of molasses and 2.5% of soybean residual oil was carried out in a bioreactor 1.2 L at 28 °C for 144 h under 200 rpm. The operation in the prototype was conducted without addition of the biosurfactant and with addition of the biosurfactant in crude form using a central rotational compound design (DCCR), where the influence of the independent variables of effluent flow and biosurfactant flow on the response efficiency variable of separation. According to the results obtained, it was observed that the use of the biosurfactant increased from 40% to 94% the efficiency of the DAF process. The best result was 92% removal in the condition containing biosurfactant flow rate of 0.52 L/min and the effluent flow rate of 3.80 L/min. It is concluded that the use of biosurfactants as auxiliaries in the flotation is a promising alternative in the reduction of the pollution caused by the oily waters generated in the industrial environment.