Anaerobic digestion for CH4 recovery in wastewater treatment has been carried out with different strategies to increase process efficiency, among which co-digestion and the two-stage process can be highlighted. In this context, this study aimed at evaluating the co-digestion of cheese whey and glycerol in a two-stage process using fluidized bed reactors, verifying the effect of increasing the organic loading rate (OLR) (2–20 g-COD.L−1.d−1) and temperature (thermophilic and mesophilic) in the second stage methanogenic reactor. The mesophilic methanogenic reactor (R-Meso) (mean temperature of 22 °C) was more tolerant to high OLR and its best performance was at 20 g-COD.L−1.d−1, resulting in methane yield (MY) and methane production (MPR) of 273 mL-CH4.g-COD−1 and 5.8 L-CH4.L−1.d−1 (with 67% of CH4), respectively. Through 16S rRNA gene massive sequencing analysis, a greater diversity of microorganisms was identified in R-Meso than in R-Thermo (second stage methanogenic reactor, 55 °C). Firmicutes was the phyla with higher relative abundance in R-Thermo, while in R-Meso the most abundant ones were Proteobacteria and Bacteroidetes. Regarding the Archaea domain, a predominance of hydrogenotrophic microorganisms could be observed, being the genera Methanothermobacter and Methanobacterium the most abundant in R-Thermo and R-Meso, respectively. The two-stage system composed with a thermophilic acidogenic reactor + R-Meso was more adequate for the co-digestion of cheese whey and glycerol than the single-stage process, promoting increases of up to 47% in the energetic yield (10.3 kJ.kg-COD−1) and 14% in organic matter removal (90.5%).