Deep geological repositories (DGRs) stand out as one of the optimal options for managing high-level radioactive waste (HLW) such as uranium (U) in the near future. Here, we provide novel insights into microbial behavior in the DGR bentonite barrier, addressing potential worst-case scenarios such as waste leakage (e.g., U) and groundwater infiltration of electron rich donors in the bentonite. After a three-year anaerobic incubation, Illumina sequencing results revealed a bacterial diversity dominated by anaerobic and spore-forming micro-organisms mainly from the phylum Firmicutes. Highly U tolerant and viable bacterial isolates from the genera Peribacillus, Bacillus, and some SRB such as Desulfovibrio and Desulfosporosinus, were enriched from U-amended bentonite. The results obtained by XPS and XRD showed that U was present as U(VI) and as U(IV) species. Regarding U(VI), we have identified biogenic U(VI) phosphates, U(UO2)·(PO4)2, located in the inner part of the bacterial cell membranes in addition to U(VI)-adsorbed to clays such as montmorillonite. Biogenic U(IV) species as uraninite may be produced as result of bacterial enzymatic U(VI) reduction. These findings suggest that under electron donor-rich water-saturation conditions, bentonite microbial community can control U speciation, immobilizing it, and thus enhancing future DGR safety if container rupture and waste leakage occurs. Environmental ImplicationsEnsuring future nuclear waste repository safety requires consideration of physicochemical and microbiological factors. This study provides novel insights into microbial behavior in bentonite barriers, addressing worst-case scenarios like waste leakage (e.g., uranium) and groundwater infiltration. We present evidence of the enrichment of anaerobic and spore-forming microbes and viable sulfate-reducing bacteria (SRB) upon U-amended bentonite after a 3-year incubation. Furthermore, U speciation was affected by both biotic and abiotic processes, leading to its immobilization as U(VI) phosphates, uraninite, in addition to its sorption onto bentonite minerals. This implies that the U-immobilization within this barrier could positively enhance the safety performance of future DGRs.