The shift of microbial communities during a transition from mesophilic anaerobic digestion (MAD) to thermophilic anaerobic digestion (TAD) was characterized in two treatments. One treatment was inoculated with sludge and the other was inoculated with manure. In this study, methane was produced both in MAD and TAD, but TAD has slightly more methane produced than MAD. A broad phylogenetic spectrum of bacterial, archaeal, and fungal taxa at thermophilic conditions was detected. Coprothermobacter, Bacillus, Haloplasma, Clostridiisalibacter, Methanobacterium, Methanothermobacter, Saccharomycetales, Candida, Alternaria, Cladosporium, and Penicillium were found almost exclusively in TAD, suggesting their adaptation to thermophilic conditions and ecological roles in digesting the organic compounds. The characterization of the lesser-known fungal community revealed that fungi probably constituted an important portion of the overall community within TAD and contributed to this process by degrading complex organic compounds. The shift of the microbial communities between MAD and TAD implied that temperature drastically affected the microbial diversity in anaerobic digestion. In addition, the difference in microbial communities between sludge and manure indicated that different source of inoculum also affected the microbial diversity and community.