Botryosphaeriaceae species are among the most common fungi isolated from grapevine (Vitis vinifera) cankers in Australia. Thirty-eight isolates comprising eight Botryosphaeriaceae species isolated from grapevine cankers throughout New South Wales and South Australia were used in a pathogenicity study on mature wood of 15-year-old Chardonnay grapevines. Experiments showed that all eight species were able to infect grapevines under field conditions causing vascular discoloration and/or staining of the wood (lesions). However, differences in pathogenicity were evident among strains and species (P < 0.001). Neofusicoccum parvum and Lasiodiplodia theobromae were the most pathogenic based on lesion length, followed by Neofusicoccum australe, Botryosphaeria dothidea and Diplodia mutila and thereafter by Dothiorella viticola, Dothiorella iberica and Diplodia seriata, which were the least pathogenic. Growth rates also differed significantly among species depending on temperature (P < 0.001). Lasiodiplodia theobromae and B. dothidea grew optimally between 29 and 30 °C, Diplodia and Neofusicoccum spp. between 25 and 27 °C, and Dothiorella spp. at temperatures between 22 and 24 °C. Temperature-growth relationships correlated well with previous data on the prevalence and distribution of Botryosphaeriaceae species in Australian vineyards. Due to their pathogenicity, prevalence, distribution and tolerance to a wide range of environmental conditions, the Botryosphaeriaceae pose a significant threat to the Australian wine industry.