We present the discovery of a Middle Pleistocene fossil assemblage at Los Villares locality (Ruidera, Ciudad Real, Castilla-La Mancha), which has possible evidence of associated human activity. The potential of the site has been evaluated through multidisciplinary research including taxonomy, anatomy, deep learning, and direct dating of fossil remains. A surface study carried out in 2017, over a very limited area (2 m2) on the slope of one of the Ruidera lakes led to the discovery of more than 50 fossil specimens, including cranial (mainly teeth) and postcranial remains. This rich assemblage is dominated by the remains of Caprinae, although the presence of some small or medium carnivore remains also stands out. The identification of a cut mark, tested with Convolutional Neural Networks, suggests the presence of human activity within the bone assemblage. Several fossils were directly dated using a multi-technique approach involving radiocarbon, U-Th, and ESR methods. The results constrain the fossil assemblage to between 300 ka and 400 ka, positioning Los Villares as one of the first Middle Pleistocene localities identified in the Upper Guadiana basin, on the Southern edge of the Southern Iberian Plateau. These promising initial results show the great potential of the site to contrib-ute to filling a gap of knowledge in the Pleistocene archaeo-paleontological record of the Iberian Peninsula. Nevertheless, we also acknowledge the need for systematic excavations in the future, not only to obtain a better idea of the lateral and stratigraphic extension of the fossil assemblage and its complete taxonomic composition, but also to confirm the human presence at the site.