Botryosphaeria dieback (BD) is a serious disease of grapevines causing considerable economic loss worldwide including in Australia. The disease is caused by several Botryosphaeriaceae species that result in symptoms such as cankers, dieback and in latter stages the death of vines. In other countries, the disease is also associated with foliar symptoms, however, these have not been reported in Australian wine grape cultivars to date. Many foliar symptoms are usually associated with the production of phytotoxic metabolites produced by the pathogen and that are translocated into the leaves. The role of secondary metabolites in pathogenicity still required further study. Thus, investigating the production of phytotoxins from Botryosphaeriaceae species in Australian vineyards could result in the role of phytotoxins in the pathogenicity being elucidated. This may also assist in field diagnosis and the development of more effective control strategies for BD in Australian vineyards. The production of phytotoxins from the most widespread and virulent Botryosphaeriaceae species associated with BD in Australia were investigated and included three isolates each of Neofusicoccum parvum, Dothiorella iberica, Spencermartinsia viticola and four isolates of Diplodia seriata. All isolates were cultured in Czapek Dox broth and their culture filtrates extracted at different pH’s with ethyl acetate. All filtrates and organic extracts were assayed for phytotoxicity using grapevine leaves and tomato seedlings. The results showed that the degree of toxicity differed between isolates, species and assay conditions. The secondary metabolites from the organic extracts produced from the 13 culture filtrates were screened using thin layer chromatography (TLC). From the bioassay and TLC results, D. seriata (H141a), Do. iberica (L5) and S. viticola (L19) were further selected, and the isolation, chemical and biological characterisation of their secondary metabolites is in progress.