Myrtle rust (MR) is a severe fungal disease, which threatens Myrtaceae plant species, including commercially and ecologically important timber, paperbark, and tea tree species in Australia. The causative agent, Austropuccinia psidii, can infect 393 native Australian Myrtaceae species. At least three species are critically endangered, and several species may become extinct within one generation. We aim to investigate differences in fungal diversity between MR-susceptible and MR-resistant trees, and to test the effectiveness of fungi against A. psidii and other plant pathogens. We have isolated both leaf surface and endophytic fungi from MR-susceptible and MR-resistant Melaleuca quinquenervia trees using standardized leaf washing and surface sterilization techniques, respectively. Isolated fungi were cultured and identified through ITS sequencing, and the most prominent genera were Penicillium and Cladosporium. Notably, we observed significant differences in the fungal diversity between MR-susceptible and MR-resistant trees. In initial screening, we tested the inhibitory activity of isolated fungi against the gummy stem blight pathogen Stagonosporopsis cucurbitacearum and A. psidii through co-culture and in vitro assays, respectively. A significant proportion of the isolated fungi were active against S. cucurbitacearum and inhibited the germination of A. psidii. These findings suggest that leaf-associated fungi may play a role in natural disease resistance and could serve as a basis for developing targeted biocontrol strategies. In the future, we plan to further explore the metabolites produced by the identified potential fungi which may inhibit A. psidii. This knowledge could inform the development of effective, environmentally friendly, and sustainable biocontrol strategies to protect threatened Myrtaceae species.