Fungi belonging to the family Botryosphaeriaceae have been implicated in significant economic loss in many horticultural crops worldwide. These fungal pathogens pose an emerging risk to the productivity of the Australian walnut industry, although which species of Botryosphaeriaceae are present is unknown. To address this knowledge gap, walnut tissues were collected from 14 orchards representing the major walnut-growing regions of Australia, and fungal isolations performed. Botryosphaeriaceae species were recovered from both symptomatic and asymptomatic samples from nine of the orchards. DNA sequencing confirmed the presence of five Botryosphaeriaceae species, namely Diplodia seriata, Dothiorella omnivora, Neofusicoccum parvum, N. macroclavatum and Spencermartinsia viticola. Of the 106 Botryosphaeriaceae isolates recovered, D. seriata and N. parvum were the most prevalent species. Pathogenicity studies using detached stems indicated that Neofusicoccum spp. were the most virulent followed by D. seriata. This is the first systematic study on the incidence, identification and pathogenicity of species of Botryosphaeriaceae from walnut orchards in Australia.