Trypanosomes are an important group of flagellate blood parasites in both livestock and humans, yet Australian wildlife trypanosomes remain largely understudied. Recent investigations into Australian trypanosomes in native marsupials observed Trypanosoma copemani isolated from the brush-tailed bettong (Bettongia pencillata) invading mammalian cells. Few trypanosomes have been observed infecting mammalian cells, with T. cruzi, the causative agent of Chagas disease in humans being the most widely studied. Additionally, T. sp. H25 isolated from a number of marsupials in Australia was found to be genetically similar to T. cruzi. These findings have led to a growing focus on the importance of understanding the biodiversity and life histories of trypanosomes occurring naturally in Australia. High resolution imaging tools, including scanning and transmission electron microscopy have assisted in documenting parasite biodiversity by investigating the ultrastructure of various morphological forms of recently described trypanosomes in Australia. While using a combination of in vitro cell culture methods, and live cell and fluorescence imaging have provided new insights into the host-parasite relationships and life histories of various Australian wildlife trypanosomes. The most significant of these concern their pathogenicity in vulnerable Australian wildlife, and the identification of possible biosecurity hazards associated with the transmission of T. cruzi by Australian marsupials.