The Chalcolithic levels of El Portalón de Cueva Mayor (Atapuerca, Burgos, Spain) offer a good opportunity to test whether the small-mammal contents of different archaeo-stratigraphical units may be useful to characterize them as independent entities. With that purpose, we studied representative samples of small-mammal remains from the two main contexts identified: the Early Chalcolithic (EC) funerary context and the Late Chalcolithic (LC) habitat/stabling context, with the latter comprising three different archaeological units according to their origin, namely prepared floors, activity floors and stabling surfaces or fumiers. Following the distribution of taxa in their respective contexts, we performed several statistical tests to check for significant discrepancies between archaeological units. The exclusive presence of certain taxa, together with the statistical difference in relative taxonomic ratios, points to the integrity and unpolluted condition of the EC context. The interspersed arrangement of the different LC context's units made them prone to inter-pollution as they are not statistically different. The unexpected presence of Pliomys lenki and Chionomys nivalis in the prepared floors evidences their Upper Pleistocene allochthonous origin. The EC levels of El Portalón contribute the first Holocene records of nine taxa in the Sierra de Atapuerca. An environment dominated by woodland, shrubland and wet meadows, with moderate presence of grassland, inland wetlands and rocky areas, is inferred from the small-mammal association of the EC levels.