Biosurfactants are a group of environmentally friendly amphiphilic molecules that are applicable in numerous industries as essential biotechnology products, such as food production, cleaning products, pharmacology, cosmetics, pesticides, textiles and oil and gas fields. In this sense, and knowing the potential of these biomolecules, the aim of this work was to produce a biosurfactant, characterize it regarding its chemical and surfactant properties and investigate its potential in the removal of contaminants and in the cleaning of cotton fabrics. The biosurfactant was initially obtained from the cultivation of the microorganism Candida glabrata UCP 1002 in medium containing distilled water with 2.5% residual frying oil, 2.5% molasses and 2.5% corn steep liquor agitated at 200 rpm for 144 h. The biosurfactant reduced the surface tension of water from 72 to 29 mN/m. The toxicity potential of the biosurfactant was evaluated using Tenebrio molitor larvae and demonstrated non-toxicity. The biosurfactant was applied as a degreaser of engine oil on cotton fabric, and showed 83% (2× CMC), 74% (1× CMC) and 78% (1/2× CMC) oil removal. Therefore, the biosurfactant produced in this work has promising surfactant and emulsifying properties with potential for application in various industrial segments.