<p>Ferruginous conditions in the Precambrian oceans gave rise to ancient iron formations that contain the oldest recognized traces of life in the form of mineral biosignatures and isotope imprints. However, these biogeochemical archives lack clear constraints on microbial diagenesis prior to lithification as metabolic functions of the primeval subsurface biosphere remain elusive. Integrating metagenomics with mineralogy, we show that microbial populations in ferruginous Lake Towuti mediate authigenic minerals (magnetite, millerite, siderite, vivianite) consistent with dissimilatory respiration types predicted from marker genes in the shallow subsurface. Syntrophic fermentation sustained long-term mineralization of reactive iron and organic carbon substrates in the deep biosphere. Ferric to ferrous diagenetic redistribution via pore water promoted isotope fractionation during burial, producing negative and positive δ⁵⁶Fe signatures in end-members siderite and vivianite, and poorly reactive phases, respectively. Trophic fractionation of organic carbon during remineralization produced differential δ¹³C signatures in siderites, following mass balance between methane and biogenic dissolved inorganic carbon. Persistent archaea exhibited means of survival on minimal organic carbon, namely an enhanced C1 backbone metabolism grafted onto a mixotrophic Wood-Ljungdahl pathway. Metagenomic predictions thereby elucidated mineralization metabolic pathways and their target substrates responsible for biosignatures imprinted during early (20 ka) and long-term (100 ka) diagenesis.</p>