Bentonite is currently proposed as a potential backfill material for sealing high-level radioactive waste in underground repositories due to its low hydraulic conductivity, self-sealing ability and high adsorption capability. However, saline pore waters, high temperatures and the influence of microbes may cause mineralogical changes and affect the long-term performance of the bentonite barrier system. In this study, long-term static batch experiments were carried out at 25 °C and 90 °C for one and two years using two different industrial bentonites (SD80 from Greece, B36 from Slovakia) and two types of aqueous solutions, which simulated (a) Opalinus clay pore water with a salinity of 19 g·L−1, and (b) diluted cap rock solution with a salinity of 155 g·L−1. The bentonites were prepared with and without organic substrates to study the microbial community and their potential influence on bentonite mineralogy. Smectite alteration was dominated by metal ion substitutions, changes in layer charge and delamination during water–clay interaction. The degree of smectite alteration and changes in the microbial diversity depended largely on the respective bentonite and the experimental conditions. Thus, the low charged SD80 with 17% tetrahedral charge showed nearly no structural change in either of the aqueous solutions, whereas B36 as a medium charged smectite with 56% tetrahedral charge became more beidellitic with increasing temperature when reacted in the diluted cap rock solution. Based on these experiments, the alteration of the smectite is mainly attributed to the nature of the bentonite, pore water chemistry and temperature. A significant microbial influence on the here analyzed parameters was not observed within the two years of experimentation. However, as the detected genera are known to potentially influence geochemical processes, microbial-driven alteration occurring over longer time periods cannot be ruled out if organic nutrients are available at appropriate concentrations.