Residual oil from babassu (Orbignya sp.), a low-cost raw material, was used in the enzymatic esterification for biodiesel production, using lipase B from Candida antarctica (Novozym® 435) and ethanol. For the first time in the literature, residual babassu oil and Novozym® 435 are being investigated to obtain biodiesel. In this communication, response surface methodology (RSM) and a central composite design (CCD) were used to optimize the esterification and study the effects of four factors (molar ratio (1:1–1:16, free fatty acids (FFAs) /alcohol), temperature (30–50 °C), biocatalyst content (0.05–0.15 g) and reaction time (2–6 h)) in the conversion into fatty acid ethyl esters. Under optimized conditions (1:18 molar ratio (FFAs/alcohol), 0.14 g of Novozym® 435, 48 °C and 4 h), the conversion into ethyl esters was 96.8%. It was found that after 10 consecutive cycles of esterification under optimal conditions, Novozym® 435 showed a maximum loss of activity of 5.8%, suggesting a very small change in the support/enzyme ratio proved by Fourier Transform Infrared (FTIR) spectroscopy and insignificant changes in the surface of Novozym® 435 proved by scanning electron microscopy (SEM) after the 10 consecutive cycles of esterification.