This work presents experimental results on alkali and enzymatic catalysis of corn oil into biodiesel with an optimization of operating conditions and further experiments on enzyme reuse. A comparison of the alkali-catalyzed methanolysis and ethanolysis of corn oil is done, followed by the study of the enzymatic-catalyzed ethanolysis using the alcohol at different concentrations (ethanol absolute, 96%, and 70%, v/v). Results show that the best operating conditions for biodiesel production using absolute ethanol (containing no water) as reagent are an oil/alcohol molar ratio of 1:6, a catalyst/oil weight percentage of 2.8 wt %, a reaction time of 12 h, and a reaction temperature of 35 degrees C. For these conditions it was possible to obtain a reaction yield of 98.95 wt % with a fatty acid ethyl esters (FAEE) content of 69.2 wt %, with linoleate (C18:2) and oleate (C18:1) being the most significant esters (with relative percentages of 42.97 wt % and 22.54 wt %, respectively). Regarding the evaluation of the enzyme activity loss during reaction, it was concluded that under these conditions it is possible to reuse the enzyme four times after which there was a significant loss of the biodiesel quality according to the EN 14214:2009 standard.