Incidence of diseases associated with species in the fungal family Botryosphaeriaceae are increasing, and becoming a major economic concern in several horticultural tree crops worldwide. Species of Botryosphaeriaceae act in many ways but most commonly as endophytes, causing latent infections or are serious outright pathogens. In most cases, the fungal organism enters the host through wounds or natural openings. The resulting symptoms include branch and trunk cankers, dark lesions of the tissue beneath the bark of stems, leaf and stem blight, fruit rot, and in severe cases tree death. Due to the wide host range of many Botryosphaeriaceae, it is hypothesised that there is strong cross-pathogenicity of Botryosphaeriaceae isolates across multiple host species. In this study, we developed a rapid bioassay to compare host specificity and pathogenic variation of fungal isolates of species in the family Botryosphaeriaceae on different tree crops including macadamia and blueberry. Using a detached leaf inoculated at the petiole with the test isolate in a microcosm and maintained under moist, semi-sterile conditions at 25°C and 100% relative humidity, host susceptibility of blueberry and macadamia cultivars to species of Botryosphaeriaceae was determined. From preliminary studies it has been observed that blueberry is highly susceptible to isolates of Botryosphaeriaceae isolated from different plant hosts with macadamia proving to be far more resilient. This pilot study revealed that the detached leaf bioassay reflects in planta and stem inoculation assays and is rapid for quantitative analysis of virulence and aggressiveness of species of Botryosphaeriaceae on different tree hosts.