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The wastewater from the biodiesel industry is an environmental problem, and from a sanitation resources perspective, the anaerobic sequencing batch reactor (ASBR) is an interesting alternative for wastewater treatment. A better understanding of ASBR operation behavior under the progressive increase of the organic loading rate (OLR) is crucial for upscaling. The objective of this study was to monitor an ASBR operating with an OLR ranging from 1.3 to 9.3 kgCOD m−3 d−1. The average chemical oxygen demand (COD) removal efficiencies of the ASBR were 52, 41, 47, and 11% for phases 1, 2, 3, and 4, respectively. The apparent kinetic coefficient, i.e., the rate of degradation of organic matter, was between 0.10 and 1.80 h−1, considering the kinetic model that considers the residual substrate concentration, which was the one that best fit the obtained data. The progressive increase in applied OLR modified the microbial biomass diversity, which in turn influenced the degradation kinetics of the organic matter. In addition, the values of the applied OLR of 5.1 kgCOD m−3 d−1 and a food to microorganism ratio (F/M) of 0.6 kgCOD kgVSS−1 d−1 were shown to be limiting values that promoted the overload of ASBR.