Anaerobic digestion has proven to be an economically and environmentally viable way of disposing of industrial, agro-industrial and domestic wastes due to the use of biogas for energy generation (biomethane). In this study, a by-product of biodiesel production (crude glycerol) was used as a substrate infor biological treatment in a sludge blanket anaerobic reactor (UASB) with hydraulic retention time (HRT) of 24 h and different organic loading rate (OLR) of 0.52, 1.09 and 2.02 kgCOD m−3 d−1. Under these operating conditions (140 days), the average removal efficiency of 74% (total COD - chemical oxygen demand) was achieved as well as effluent concentration raging between 100 and 500 mgO2 L−1 and biogas production of 5.1 L d−1. The performance of the UASB reactor was evaluated in phases 1 and 2, during which average organic matter removal efficiencies of 64% (phase 1) and 81% (phase 2) were achieved, corresponding to effluent mean values of 189 ± 57 mg O₂ L−1 (phase 1) and 211 ± 110 mg O₂ L−1 (phase 2). Moreover, no accumulation of total volatile acids (TVA) was observed, with the maximum value reaching 200 mg L⁻1 (as acetic acid). Sodium bicarbonate (0.5 and 1.0 g NaHCO₃ L−1) was also required to buffer the effluent and maintain alkalinity in the anaerobic reactor. Therefore, the system was shown to be effective in treating the residue generated from the biodiesel production process using waste cooking oil. This alternative would be interesting for application on a larger scale as a source of biogas production.