Middens (nests and caches) of Late Pleistocene arctic ground squirrels (Urocitellus parryii) that are preserved in the permafrost of Beringia archive valuable paleoecological data. Arctic ground squirrels selectively include the plant material placed in middens. To account for this selectivity bias, we used a multi-proxy approach that includes ancient DNA (aDNA) and macro- and microfossil analyses. Here, we provide insight into Pleistocene vegetation conditions using macrofossils, pollen, phytoliths and non-pollen palynomorphs, and aDNA collected from one such midden from the Yukon Territory (Canada), which was formed between 30,740 and 30,380 cal yr BP. aDNA confirmed the midden was constructed by U. parryii. We recovered 39 vascular plant and bryophyte genera and 68 fungal genera from the midden samples. Grass and other herbaceous families dominated vegetation assemblages according to all proxies. aDNA data yielded several records of vascular plants that are outside their current biogeographic range, while some of the recovered fungi yielded additional evidence for local occurrence of Picea trees during glacial conditions. We propose that future work on fossil middens should combine the study of macro- and microfossils with aDNA analysis to get the most out of these environmental archives.