The study aimed to utilize MoO3 catalysts, produced on a pilot scale via combustion reaction, to produce biodiesel from residual oil. Optimization of the process was conducted using a 23 experimental design. Structural characterization of the catalysts was performed through X-ray diffraction, fluorescence, Raman spectroscopy, and particle size distribution analyses. At the same time, thermal properties were examined via thermogravimetry and differential thermal analysis. Catalytic performance was assessed following process optimization. α-MoO3 exhibited a monophasic structure with orthorhombic phase, whereas α/h-MoO3 showed a biphasic structure. α-MoO3 had a larger crystallite size and higher crystallinity, with thermal stability observed up to certain temperatures. X-ray fluorescence confirmed molybdenum oxide predominance in the catalysts, with traces of iron oxide. Particle size distribution analyses revealed polymodal distributions attributed to structural differences. Both catalysts demonstrated activity under all conditions tested, with ester conversions ranging from 93% to 99%. The single-phase catalyst had a long life cycle and was reusable for six biodiesel production cycles. The experimental design proved to be predictive and significant, with the type of catalyst being the most influential variable. Optimal conditions included α-MoO3 catalyst, oil/alcohol ratio of 1/15, and a reaction time of 60 min, resulting in high biodiesel conversion rates and showcasing the viability of MoO3 catalysts in residual oil biodiesel production.