Context Establishing appropriate faunal baselines is critical for understanding and abating biodiversity declines. However, baselines can be highly reliant on historical records that come from already disturbed ecosystems. This is exemplified in the Murray-Darling Depression bioregion of Australia, where European settlement (and accompanying marked land-management changes and the introduction of many species) triggered rapid declines and losses of native species, often before their documentation. Aims We aim to establish the mammal fauna present when Europeans settled the Murray Mallee and Murray-Darling Depression bioregion and determine the extent of mammal loss since European settlement. Methods We describe a dated vertebrate assemblage from Light's Roost in the lower Murray Mallee region of South Australia. We compare our data with those of modern fauna surveys and historical records to document the extent of change in the mammal fauna since European settlement. Key results Radiocarbon ages showed that the assemblage was accumulating, at a minimum, within an interval from 1900 to 1300 years ago. Since this time, the Murray-Darling Depression has lost half of its flightless terrestrial mammals. Species lost include the mulgara (Dasycercus blythi/cristicauda), which places this taxon within only 40km of Lake Alexandrina, the hitherto-disputed type locality for D. cristicauda. Fossils provided the principal evidence for nearly half of the Murray Mallee fauna and over three-quarters of the fauna are represented in the fossil record. Conclusions Late Holocene assemblages provide important archives of species biogeography and diversity. Our revised faunal baseline indicated that the pre-European fauna of the Murray-Darling Depression was more diverse than hitherto understood and its reduction appears largely caused by the impacts of European settlement. Implications Baselines for species distributions derived from historical records and modern faunal surveys are likely to be incomplete and warrant revision, particularly for smaller and more cryptic species. Deficiencies in regional records mask the extent of mammal declines caused by European colonisation and associated agricultural practices, and thus vulnerability to anthropogenic disturbance.