During the last glacial period, rapidly changing environments posed substantial challenges to Neanderthal populations in Europe. Southern continental regions, such as Iberia, have been proposed as important climatic “buffer” zones during glacial phases. Contextualising the climatic and ecological conditions Neanderthals faced is relevant to interpreting their resilience. However, records of the environments and ecosystems they exploited across Iberia exhibit temporal and spatial gaps in coverage. Here we provide new evidence for palaeotemperatures, vegetation structure, and prey herbivore ecology during the late Pleistocene (MIS 5–3) in northern Spain, by applying multiple stable isotope tracers (δ18O, δ13C, δ15N, δ34S) to herbivore skeletal remains associated with Neanderthal occupations at Axlor Cave, Bizkaia. The results show little change over time and indicate stable climatic conditions and ecosystems across different occupations. Large within-layer isotopic variability in nitrogen and sulphur suggests the presence of a mosaic environment and a variety of isotopic ecotones that were exploited by Neanderthals and their prey. We implement a combination of carbonate and phosphate δ18O measurements to estimate palaeotemperatures using a cost-effective workflow. We show that the targeted use of phosphate δ18O measurements to anchor summer peak and winter trough areas enables high-precision seasonal palaeoclimatic reconstructions.