Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.