The rapid development of biotechnology and the increase in environmental awareness among producers and consumers have placed biological products in a preferential position on the market. The physiochemical properties of biosurfactants make these natural compounds an attractive option with potential use in a variety of industrial and biotechnological applications as additives in foods, cosmetics and detergents. Thus, the aim of the present study was to select the better biosurfactant-producing microorganism between two species of Candida (C. guilliermondii UCP0992 and C. lipolytica UCP0998) grown in media with different sources of carbon (glucose, sugarcane molasses and waste frying oil) and nitrogen (yeast extract, urea and corn steep liquor). After the selection of the microorganism and production medium and biosurfactant production were analyzed. The properties of the biopolymer were then studied in terms of toxicity, using the brine shrimp Artemia salina, vegetable seeds and onion (Allium cepa) as bioindicators. Gas chromatography was performed for the structural characterization of the biomolecule and the determination of its physicochemical composition. Candida guilliermodii grown in a medium with 5.0% molasses, 5.0% corn steep liquor and 5.0% waste frying oil was selected for the production of biosurfactant, with a reduction in surface tension to 28.6 mN/m, a yield of 21 g/l and a critical micelle concentration of 0.7 g/l. The isolated biosurfactant was not toxic to Artemia salina, or the vegetable seeds tested. The biosurfactant had a glycolipid nature. Based on the present results, the novel biosurfactant produced herein has potential application in industrys.