Conilurus penicillatus, Mesembriomys macrurus and M. gouldii have undergone significant contraction in distribution and abundance since European settlement. The Kimberley region is one where they co-occurred and the project’s aim was to determine niche separation within a restricted area of the Mitchell Plateau. Trapping at ground level, observations and radio telemetry at nine sites in vine thicket and open eucalypt forest were used to describe habitat preference, use and diet. Vegetation requirements were characterized using PATN analysis. Vegetation analysis showed little overlap in habitat preference. Mesembriomys gouldii preferred relatively stable open forest with a well-developed shrub layer, nesting in tree hollows and palm fronds. Mesembriomys macrurus selected several habitats, especially the edges between vine thicket and open forest, nesting in hollows in large eucalypt trees. Conilurus penicillatus occupied the greatest variety of habitats, nesting in eucalypts and ground-level sites. C. penicillatus was the most frequently trapped and found in both trees and on the ground. M. gouldii was predominantly arboreal while M. macrurus was mostly arboreal but also spent time on the ground. While there is substantial overlap in diet, niches were identified: M. gouldii a granivore/frugivore, eating seeds almost exclusively; M. macrurus an omnivore with a diet of seeds, fruits, flowers, termites and other invertebrates; and C. penicillatus a generalist whose diet included monocot and dicot leaves, seeds, flowers, fruits and arthropods. Landscape-wide changes in northern Australia since European settlement may have reduced the availability of the vital and differing resources required by these tree-rat species.