The Balkan Peninsula is considered to have acted as a glacial refugium as well as a biogeographic crossroads during the Pleistocene, playing an important role in the survival of biota and population dynamics through time. Furthermore, rather than being a homogeneous habitat, it is thought to have hosted a number of diverse, isolated “refugia within refugia,” providing suitable conditions for the persistence of different plant and animal species, as well as, potentially, Pleistocene hominins. We present the first palynological and charcoal record, although discontinuous, from the middle Pleistocene archaeological site Marathousa 1 (MAR-1; Megalopolis Basin) to reconstruct the local environment along with the first multispecies record of coprophilous fungal spores from Greece to track herbivore activity during the Marine Isotope Stage (MIS) 12. Our data show that during the early and late MIS 12, when the forest cover substantially decreased, mesophilous trees and aquatic vegetation persisted, reflecting wetter and milder conditions at MAR-1. Herbivore presence is documented by ∼473 ka, while its intensification coincides with increased vegetation biomass and local fire activity during the late MIS 12. Our findings suggest that MAR-1 likely served as a glacial refugium for middle Pleistocene hominins, providing essential resources for their survival during MIS 12.