© 2022 by the authors.In this study, predominant bacterial and archaeal populations and their roles during anaerobic mono-digestion of food waste (FW) and co-digestion of FW with straw pellets (SP) at thermophilic temperature (53 ± 1 °C) were assessed by Next Generation Sequencing (NGS) analysis at organic loading rates (OLRs) of 3.0 and 7.0 gVS/L/d. Depending on the seed; results revealed that Firmicutes, Bacteroidetes, and Proteobacteria were, respectively the most prevalent bacterial phyla at both OLRs investigated. On the other hand, Euryarchaeota was dominated by methanogens playing crucial role in biogas production and correlated mainly with the activities of Methanobacteria and Methanomicrobia at class level. Acetoclastic Methanosaetae was the predominant genus at OLR = 3.0 gVS/L/d; however, shared the same predominance with hydrogenotrophic methanogens Methanospirillium at the highest OLR. Although no clear effect in response to straw addition at OLR of 3.0 gVS/L/d could be seen in terms of methanogenic archaea at genus level, hydrogenotrophic methanogens revealed some shift from Methanobacterium to Methanospirillium at higher OLR. Nevertheless, no prominent microbial shift in the presence of wheat straw at increased OLR was likely due to adapted inoculation at start-up which was also demonstrated by relatively stable biogas yields during co-digestion.