The Batagay megaslump, a permafrost thaw feature in north-eastern Siberia, provides access to ancient permafrost up to ∼650 kyr old. We aimed to assess the permafrost-locked organic matter (OM) quality and to deduce palaeo-environmental information on glacial–interglacial timescales. We sampled five stratigraphic units exposed on the 55 m high slump headwall and analysed lipid biomarkers (alkanes, fatty acids and alcohols). Our findings revealed similar biogeochemical signatures for the glacial periods: the lower ice complex (Marine Isotope Stage (MIS) 16 or earlier), the lower sand unit (sometime between MIS 16–6) and the upper ice complex (MIS 4–2). The OM in these units has a terrestrial character, and microbial activity was likely limited. Contrarily, the n-alkane and fatty acid distributions differed for the units from interglacial periods: the woody layer (MIS 5), separating the lower sand unit and the upper ice complex, and the Holocene cover (MIS 1), on top of the upper ice complex. The woody layer, marking a permafrost degradation disconformity, contained markers of terrestrial origin (sterols) and high microbial decomposition (iso- and anteiso-fatty acids). In the Holocene cover, biomarkers pointed to wet depositional conditions and we identified branched and cyclic alkanes, which are likely of microbial origin. Higher OM decomposition characterised the interglacial periods. As climate warming will continue permafrost degradation in the Batagay megaslump and in other areas, large amounts of deeply buried ancient OM with variable composition and degradability are mobilised, likely significantly enhancing greenhouse gas emissions from permafrost regions.