Methanogenic populations were investigated in subsaline Laguna Potrok Aike sediments, southern Argentina.Microbial density and activity were assessed via cell count and in situ ATP detection for the last ∼11K years. Methanogen phylogenetics highlighted species stratification throughout depth, whereas CO2 reduction was the major pathway leading to methane production. Organic substrates, characterized using pore water analysis, bulk organic fractions and saturated fatty acids, showed a clear link between sediment colonization and initial organic sources. Concentrations and δ13C compositions of methane and fatty acids provided final evidence of a microbial imprint on Holocene organic proxies in the most colonized intervals.