Pronounced glacial and interglacial climate cycles characterized northern ecosystems during the Pleistocene. Our understanding of the resultant community transformations and past ecological interactions strongly depends on the taxa found in fossil assemblages. Here, we present a shotgun metagenomic analysis of sedimentary ancient DNA (sedaDNA) to infer past ecosystem-wide biotic composition (from viruses to megaherbivores) from the Middle and Late Pleistocene at the Batagay megaslump, East Siberia. The shotgun DNA records of past vegetation composition largely agree with pollen and plant metabarcoding data from the same samples. Interglacial ecosystems at Batagay attributed to Marine Isotope Stage (MIS) 17 and MIS 7 were characterized by forested vegetation (Pinus, Betula, Alnus) and open grassland. The microbial and fungal communities indicate strong activity related to soil decomposition, especially during MIS17. The local landscape likely featured more open, herb-dominated areas, and the vegetation mosaic supported birds and small omnivorous mammals. Parts of the area were intermittently/partially flooded as suggested by the presence of water-dependent taxa. During MIS 3, the sampled ecosystems are identified as cold-temperate, periodically flooded grassland. Diverse megafauna (Mammuthus, Equus, Coelodonta) coexisted with small mammals (rodents). The MIS 2 ecosystems existed under harsher conditions, as suggested by the presence of cold-adapted herbaceous taxa. Typical Pleistocene megafauna still inhabited the area. The new approach, in which shotgun sequencing is supported by metabarcoding and pollen data, enables the investigation of community composition changes across a broad range of taxonomic groups and inferences about trophic interactions and aspects of soil microbial ecology.