Late Pleistocene deposits of Tournal Cave, southwestern France, provided several human occupations attributed to Mousterian, Aurignacian, and Magdalenian cultures. Some human remains (Homo neanderthalensis and Homo sapiens) were identified in each unit. This study presents a detailed temporal reconstruction of habitats surrounding the cave, using various proxies based on rich large mammal assemblages and related to biological activities. The levels correlated to MIS 3 and MIS 2 are characterized by alternating carnivore, Neanderthal, and Anatomically Modern Human (AMH) occupations. Faunal exploitation was mainly oriented towards Equus caballus and Rangifer tarandus, with a significant increase in the latter prey from the last Aurignacian level onwards. The results exhibit geographic and temporal variations of reindeer and horse body size. Reindeer can be used as a suitable ecological marker, as rapid changes are correlated to environmental turnover, whereas the horse presents a different pattern and is an accurate chronological estimator. The palaeoecological results indicate a major climate change between the two Aurignacian levels. The Mousterian and the first Aurignacian levels, documenting the Middle to Late Palaeolithic transition, show high indices of specific richness and diversity of large mammals in relation with a periglacial moderate cold and wet climate. The region developed a mixed landscape and displayed a non-analogue fauna with E. caballus, R. tarandus, Cervus elaphus, Megaloceros giganteus, Bison priscus, Bos primigenius, Capra caucasica praepyrenaica, Sus scrofa and Coelodonta antiquitatis for the ungulates and Ursus spelaeus, Ursus arctos, Crocuta crocuta spelaea, Panthera leo spelaea, Panthera pardus, Lynx spelaeus, Canis lupus and Vulpes vulpes for the carnivores. The breakdown occurring in the last Aurignacian level is characterized by a change in ecological settings, with a colder and drier climate and opening of the landscape, involving shifts in seasonality, plant phenology, reindeer body size and animal population densities. This impacted habitat fragmentation and geographic distribution of populations, implying various selective pressures that were reflected in human meat procurement and dispersal events. At Tournal Cave, the late Middle/early Late Palaeolithic transition is not directly marked by significant differences in terms of faunal exploitation, due to local climate and site function, but the major environmental shift recorded is delayed. The results indicate that abrupt climate oscillations during MIS 3 contributed to the decline of Neanderthal populations, notably because of habitat fragmentation. AMHs could have generated additional stresses.