In the current scenario, one of the major environmental problems is the treatment of oily water generated during industrial activities. Flotation employing the saturation of the effluent with air microbubbles is a very promising technique, due to its efficiency and control condition of physical variables, such as: size of the microbubbles, hydraulic retention time and the contaminant concentration in the effluent. The remediation process of contaminated areas has been driving the market towards the development and use of biodegradable surfactants, which act as alternative collectors in flotation and they is being able to increase even more the acceptance of this separation technology. The objective of the present study was to apply a biosurfactant of Bacillus methylotrophicus, in crude, formulated and isolated versions in a flotation chamber with induced pre-saturation, on a bench scale, in comparison with other commercial collectors for the treatment of an oily synthetic effluent. The results indicated that the commercial biosurfactant reached 90.52% removal and the chemical surfactant sodium dodecyl sulfate obtained 86.74% removal. The biosurfactant of B. methylotrophicus demonstrated ascending percentages of removal as the collector residence time increases in relation to the effluent. The crude biosurfactant reached 98.0% of removal, the formulated biosurfactant reached 98.39% of removal and the isolated biosurfactant reached 99.0% of oil separation, demonstrating the potential of application as a collector of oily contaminants in industrial processes.