Contamination by oil and its derivatives causes serious damage to the environment, motivating the development of innovative technologies for the removal of these contaminants, such as the use of biosurfactants. In the present study, the biosurfactant from Candida tropicalis UCP0996 produced in the low cost-medium formulated with molasses, residual frying oil, and corn steep liquor, was characterized and its toxicity, formulation, and application in removal and biodegradation of oil were investigated. The surface tension of the medium was reduced to 30.4 mN/m, yielding 4.11 g/L of isolated biosurfactant after 120 h. Tests under extreme environmental conditions indicated the stability of the biosurfactant. Chemical characterization by thin layer chromatography (TLC), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (1H NMR), and gas chromatography and mass spectroscopy (CG-MS) revealed the glycolipidic nature of the biosurfactant. The isolated biosurfactant showed no toxicity against the microcrustacean Artemia salina, while the properties of the formulated biosurfactant remained stable during 120 days of storage. The biosurfactant removed 66.18% of motor oil adsorbed in marine stones and dispersed 70.95% of oil in seawater. The biosurfactant was also able to increase by 70% the degradation of motor oil by seawater indigenous microorganisms, showing great potential to be applied as a commercial additive in the bioremediation of oil spills.