Alkaline transesterification of vegetable oils or animal fats (apolar substances) with a short-chain alcohol (polar substance) is the best-known chemical route for biodiesel production. However, because they are substances of different polarities, there is a need to promote contact between the phases. Biodiesel was produced from soybean oil, methanol, potassium hydroxide, and surfactants DBB 7104 and DBB 7107. In this paper we verify whether it is better to use surfactant DBB 7104 or DBB 7107, and if there is an optimum experimental condition for the surfactant. The yield in biodiesel was verified for different production conditions on a bench scale. From a fractional experimental plan (2 6-2), with a variation of temperature, time, methanol-to-oil mole ratio, weight of surfactant, type of surfactant and weight of catalyst, it was possible to perform a multivariate analysis of the process. The most influential variables in the response were the temperature and the weight of the catalyst. Surfactant DBB 7107 was considered better than the surfactant DBB 7104. The optimal conditions were 0.7% w/w of the surfactant DBB 7107, 1 g of KOH, 30 min, methanol to oil mole ratio of 6:1 and temperature equal to 33.5 °C.