Surfactants are molecules that reduce surface and interfacial tension, providing properties such as detergency, emulsification and phase dispersion. Most surfactants in use are derived from petroleum; however, interest in microbiological surfactants has increased due to their biodegradability, reduced toxicity and potential application in several areas of the industrial and environmental sectors. Therefore, in the present study, the biotechnological potential of the yeast Candida guilliermondii UCP 0992 was investigated in relation to the production of biosurfactant using different combinations of three compounds (cotton oil, sucrose and glucose). These parameters were used to select the best medium to be reproduced in a 5 L bioreactor at 30 ºC under agitation at 350 rpm and aeration at 0.5 vvm for 192 hours. The metabolic liquid produced in the bioreactor was subjected to determination of surface tension, isolation, toxicity and application in the formulation of a natural detergent for removing dirt.