Southern Europe is hypothesized to have acted as a glacial refugium for hominin populations during the Pleistocene. Of particular importance is South-East Europe, which most likely played a dual role, both as refugium and dispersal corridor, especially during the Middle Pleistocene glaciations, when drastic climatic conditions led to major sea level drops in the Aegean. However, little is known about the palaeoenvironmental conditions at the time of hominin presence in this region, making these hypotheses difficult to test. Here we analyze biomarker data and leaf wax stable isotopic compositions of the MIS 12 Lower Palaeolithic site Marathousa 1 (Megalopolis Basin, Greece) to assess the climatic conditions accompanying the time of hominin presence in the area. Our data indicate a major cooling affecting the north Mediterranean/Aegean domain during this time interval, with lowest temperatures recorded between ∼440–432 ka. The glacial peak is associated with changes in vegetation (i.e., from more forested to more open landscape), reduction of humidity and water availability (i.e., moisture depletion, increased evaporation). Hominins are present at the Marathousa 1 location at the end of this interval (434–432 ka), confirming that the Megalopolis Basin served as a refugium for hunter-gatherer groups during periods of harsh climatic conditions. Additionally, the progressive cooling is associated with an important sedimentary hiatus between ∼465–440 ka reflected in all circum-Mediterranean records (both marine and continental), indicating a regional impact of the MIS 12 glaciation over surface processes.