Grapevine trunk diseases (GTDs) are considered a serious threat to viticulture worldwide. Eutypa dieback (ED) and Botryosphaeria dieback (BD) caused by several Diatrypaceae and Botryosphaeriaceae species are the two most important trunk diseases affecting Australian vineyards to date. The ascospores and/or conidia of these pathogens are primarily spread by rain and wind and infect susceptible 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 Diatrypaceae and Botryopshaeriaceae inoculum as part of the investigations on spore dispersal patterns of ED and BD pathogens in Australian vineyards. Different DNA extraction protocols were evaluated using artificially-inoculated tapes and Burkard spore trap tapes collected from vineyards and one protocol was found efficient and gave consistent DNA yield. Two sets of multi-species primers for quantitative PCR (qPCR) were further developed to detect and quantify Diatrypaceae and Botryosphaeriaceae spores from a single environmental sample. The multi-species primers for Diatrypaceae and Botryosphaeriaceae species were designed to amplify fragments of the ITS region of the rRNA and β-tubulin genes, respectively. Specificity tests showed the two multi-species primers were highly specific to their corresponding target Diatrypaceae and Botryosphaeriaceae species (nine each) and did not amplify any of the 20 non-target species tested. The two qPCR methods were shown to amplify genomic DNA, synthetic DNA fragments (gBlocks®) and mixed DNA from environmental samples. The limit of detection of the qPCR for the Diatrypaceae and Botryosphaeriaceae species were ~20 fg and ~300 fg of genomic DNA, respectively. In summary, the two qPCR assays were developed for rapid and sensitive detection of ED and BD pathogens from environmental samples. These assays are currently being used to analyse spore trap tape samples collected from different grapevine growing regions in Australia.