The effluent production of oily water type has generated many environmental problems for several industries. 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. In this sense, the dissolved air flotation (DAF) process continues to be widely used in industries, both for water and wastewater supplies. The use of collectors is essential to improve the efficiency of the process, due to its specific characteristics that facilitate the adhesion of the particles and, consequently, the separation of the pollutants. These surface-active molecules of biological origin also have several advantages over synthetic surfactants such as higher biodegradability, higher foaming, less toxicity, better environmental compatibility, more tolerant to pH, salt, and temperature variation, and higher selectivity for metals and organic compounds and can be synthesized from renewable feedstocks. The aim of this study was to investigate a water-oil separation by DAF, with and without the addition of biosurfactant produced by Pseudomonas cepacia CCT 669 in mineral medium and formulated with 2.0% of corn steep and 3.0% of canola waste frying oil at 28°C for 60 h under 200 rpm. The experiments used to compare the effects of the addition of biosurfactant followed an experimental planning CCRD, where the response variable was the separation efficiency. Results indicated the biosurfactant added a considerable value to the process, increasing from 41.0% to 98.0% the separation efficiency, presenting potential of application as a collector of oily contaminants in the DAF process form.