In order to improve biosurfactant production byYarrowia lipolyticaIMUFRJ 50682, a factorial design was carried out. A24full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (ΔST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mNm−1ofΔST) were obtained in a medium composed of 10 g 1−1of ammonium sulfate and 0.5 g 1−1of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and aΔST of 19.5 mN m−1. The experimental design optimization enhancedΔEI by 110.7% andΔST by 108.1% in relation to the standard process.