ZnO/bentonite hybrids were obtained by the microwave-assisted hydrothermal method, in order to change the bentonite surface and improve its activity for biodiesel production. No previous treatment of the clay was used and in situ crystallization of ZnO was done by a fast single step method. A factorial design was used to optimize synthesis conditions (e.g., reaction time, solution pH, and ZnO amount). The hybrids were fully characterized to determine its structural, chemical, and morphological properties. The results demonstrated the positive contribution of the microwave-assisted hydrothermal method to the formation of ZnO/bentonite hybrids, in addition to showing that the pH was the most important parameter influencing the formation of ZnO. A pH value of 8 favored the incorporation of approximately 19.6% of ZnO onto the smectite particles even after 5 min of microwave irradiation. The ZnO/bentonite hybrid with the biggest amount of ZnO was applied as a catalyst in the transesterification reaction of soybean oil. As such, our results demonstrated an ethyl ester conversion rate higher than 73% with better performance than that of natural bentonite (44%), which represents approximately 66% of the improvement of the catalytic activity.