This study aimed to assess the use of mesquite pods hydrated mash as biomass for the growth of Saccharomyces cerevisiae UFEPEDA-1012 and Zymomonas mobilis UFEPEDA-205 and for ethanol production using a submerged fermentation. A 2³ factorial design was used to analyze the effects of the type of microorganism, time of fermentation and condition of cultivation on the ethanol production in mesquite pods mash (30 g 100 mL-1). From the obtained results the hydrated mesquite pods mash presented as a good substrate for the growth of S. cerevisiae and Z. mobilis in comparison to the standard media. The effect that most affected the ethanol production was the type of microorganism. The highest ethanol concentration (141.1 gL-1) was found when Z. mobilis was cultivated in mesquite pods mash under static condition for 36 hrs. Ethanol production by S. cerevisiae was higher (44.32 gL-1) after 18 hrs of fermentation under static condition. According to these results, the mesquite pods could be known as an alternative substrate to be used for biotechnological purposes, mainly for ethanol production.