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Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physico-chemistry are still not well understood due to the complexity of interactions between surface and subsurface. Within the framework of Hainich (north-western Thuringia, central Germany) Critical Zone Exploratory of the Collaborative Research Centre AquaDiva, we used the relative abundances of phospholipid-derived fatty acids (PLFAs) to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physico-chemistry. The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O₂, Fe_t and NH₄⁺ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite-oxidizing bacteria (11Me16:0 as biomarker for <i>Nitrospira moscoviensis</i>) were high at sites with elevated O₂ concentration where groundwater recharge supplies bioavailable substrates. In anoxic groundwaters more rich in Fe_t, PLFAs abundant in sulfate-reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fe_t and HCO₃⁻ concentrations, suggesting the occurrence of active iron reduction and the possible role of fungi in meditating iron solubilization and transport in those aquifer domains. In more NH₄⁺-rich anoxic groundwaters, anammox bacteria and SRB-derived PLFAs increased with NH₄⁺ concentration, further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA- and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which showed high predominance of nitrite-oxidizing bacteria <i>Nitrospira</i>, e.g. <i>Nitrospira moscoviensis</i>, in oxic aquifer zones and of anammox bacteria in more NH₄⁺-rich anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fe_t-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.