A series of catalysts containing phosphotungstic acid (PTA) supported on periodic mesoporous silica were prepared and evaluated for the production of biodiesel from used cooking oil. The catalytic and physical properties of the prepared catalysts were investigated. Mesoporous catalyst, KIT-5 (Korea Institute of Technology 5) loaded with 26% PTA showed high activity for single step esterification, resulting in the successful elimination of a second transesterification step. The yield of fatty acid methyl esters was 93% from used (waste) cooking oil and ∼100% from pure palmitic acid. In addition, the solid acid catalyst retained a proportion of its original catalytic activity after repeated recycling steps. Thus, the prepared solid acid catalyst has the potential to be used for the production of biodiesel from feedstock that contain high amounts of free fatty acids.