We examined the habitat use of the specialised arboreal folivore, the koala, in a fragmented rural landscape. Habitat use was studied at multiple spatial scales, from individual trees to the entire landscape. Koala presence was measured through scat searches, spotting individuals on transects and GPS tracking of individuals. At the landscape level, the proportion of patches with food tree species was most important. Soil type, although important for tree nutrient and toxin levels, were relatively unimportant in koala patch use. At the individual tree level, koalas used larger trees with more shelter during the day, and more food species trees during the night. We also examined the nocturnal tree use for the influence of leaf moisture, and concentrations of nitrogen, tannins, FPCs, terpenes and fibre. Using this multiscale approach enabled a comprehensive spatial and temporal understanding of habitat use in a highly specialised species. It also enables evidence-based conservation and management decisions on this iconic species in a fragmented landscape.