<p>Lake Towuti, Indonesia, is an ancient stratified lake with ferruginous (iron-rich, sulfate-poor) anoxic bottom water conditions and a long depositional record affected by redox changes in the water column and sediments. As modern analogue of Earth’s early ferruginous oceans, it enables the study of an active microbial subsurface biosphere and its role in organic matter and iron mineralization. Combining 16S rRNA genes, cell counts, pore water geochemistry, and bulk sediment profiles from a 100-m-long core, we present the first comprehensive characterization of the deep subsurface biosphere along a one-million-year lacustrine archive. Electron acceptors in the pore water became depleted at shallow depths, resulting in a drastic decrease in cell densities in the fermentative zone, where Bathyarchaeia dominate the microbial community composition. Although alpha and beta diversity reflected initial depletion of substrates during burial, they also varied across successive lithologies, indicating that sediment composition subsequent to deposition also affects diversity. The upper sediments (0–20 mblf) sheltered a dense and diverse microbial community involved in organic matter remineralization, actively producing and converting volatile fatty acids into carbon dioxide and methane. Deeper sediments (20–70 mblf) contained low-diversity microbial communities adapted to nutrient scarcity. In contrast, deepest lacustrine sediments (70–100 mblf) contained an increased microbial diversity reflecting greater availability of organic matter of terrestrial origin. Despite Bathyarchaeia being prime constituents of the deep subsurface biosphere, increased diversity in 16S rRNA gene composition was observed in discrete sediment layers (tephra, diatom ooze, peat). This demonstrated that depositional conditions remained traceable, while stratified microbial communities drove reductive diagenesis.</p>