Colletotrichum, a genus of fungi primarily comprising plant pathogens, is responsible for significant losses to economically important plants. This genus currently accommodates around 340 species which have a wide host range; however, their diversity is not well studied on Australian native plants. This study aims to taxonomically identify and assess the pathogenicity of Colletotrichum spp. associated with Australian native plants. Over 100 Colletotrichum cultures isolated from Australian native plants were acquired from state culture collections, VPRI (Victoria) and BRIP (Queensland), and new field collections in Victoria and Queensland. Multigene phylogeny and morphological characterisation were used to identify species. Results discovered several novel species, first reports in Australia, and host range expansions. These findings highlight the substantial diversity of Colletotrichum species associated with Australian native plants, and the significant overlap between species infecting agricultural and native plants. Of particular interest was C. pyricola, first described from fruit rot of pear in New Zealand, and now reported on other fruit tree species and native plants in Australia. Pathogenicity bioassays were utilised to establish Koch’s postulates to determine whether each isolate was pathogenic on its host of origin. Additionally, cross-host pathogenicity bioassays were used to show that C. pyricola has a broader host range on a diversity of eucalypt species. These findings extend the known host range of C. pyricola from temperate and sub-tropical fruit trees to Australian native plants and demonstrates the potential risk C. pyricola may pose to both native and agricultural crops.