In the multibarrier concept for the deep geological disposal of high-level radioactive waste (HLW), bentonite is proposed as a potential barrier and buffer material for sealing the space between the steel canister containing the HLW and the surrounding host rock. In order to broaden the spectra of appropriate bentonites, we investigated the metabolic activity and diversity of naturally occurring microorganisms as well as their time-dependent evolution within the industrial B25 Bavarian bentonite under repository-relevant conditions. We conducted anaerobic microcosm experiments containing the B25 bentonite and a synthetic Opalinus Clay pore water solution, which were incubated for one year at 30 and 60 °C. Metabolic activity was only stimulated by the addition of lactate, acetate, or H2. The majority of lactate- and H2-containing microcosms at 30 °C were dominated by strictly anaerobic, sulfate-reducing, and spore-forming microorganisms. The subsequent generation of hydrogen sulfide led to the formation of iron-sulfur precipitations. Independent from the availability of substrates, thermophilic bacteria dominated microcosms that were incubated at 60 °C. However, in the respective microcosms, no significant metabolic activity occurred, and there was no change in the analyzed biogeochemical parameters. Our findings show that indigenous microorganisms of B25 bentonite evolve in a temperature- and substrate-dependent manner.