The aim of this thesis is to assess the role of diet in the extinction of Australia's iconic marsupial carnivore, the thylacine (Thylacinus cynocephalus) in Tasmania. Herein, we present two novel techniques to address fundamental questions regarding their maximum prey size and potential competition with sympatric predators. Three-dimensional computer models of the thylacine skull were used to assess their biomechanical limitations in prey size within a comparative context. This included living relatives from the family Dasyuiridae as well as a recently recovered fossil, Nimbacinus dickoni, from the family Thylacindae. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) of tissues from thylacine and potential prey species were used to assess the thylacine’s dietary composition. Furthermore, we integrate historical and recent marsupial carnivore stable isotope data to assess long-term changes in the ecosystem in response to multiple human impacts following European settlement. Our biomechanical findings support the notion that solitary thylacines were limited to hunting prey weighing less than their body mass. This concurs with our stable isotope results, which suggest medium-sized mammals were a major element of thylacine subsistence in Tasmania. Prey species may have included the common wombat (Vombatus ursinus), red-necked wallaby (Macropus rufogriseus) and Tasmanian pademelon (Thylogale billardierii). These species fall within the expected size range of prey needed to sustain this large carnivore. Competition among thylacines and sympatric marsupial carnivores in Tasmania was suggested by similarities in their prey size and trophic niche. If interspecific competition had been a major limiting factor for the small thylacine population, encounters with smaller predators were possibly reduced by occupying different habitats, as indicated by differences in predator δ13C values. The century-long change in Tasmanian devil and spotted-tailed quoll δ13C values suggests a change in vegetation in the areas typically inhabited by these species, or more likely indicates the movement of these top predators away from traditional open grassland, mosaic habitats to more densely forested habitats due to habitat loss and fragmentation. These species also displayed temporal changes in their δ15N values, which may be attributed changing prey availability or changes in the source nitrogen at the base of the food. This thesis provides a framework to test the resilience of marsupial carnivores to anthropogenic impacts in light of their biological traits and may be used to improve the conservation of large predators.