Benzothiadiazole (BTH), a salicylic acid analogue, strengthens plant defence mechanisms against a broad spectrum of pathogens. The role of pre-treatment with BTH in enhancing resistance against infection with various isolates of downy and powdery mildews (Plasmopara viticola and Erysiphe necator) was investigated in grapevine leaves. Tools were developed to better assess the defence status of the plant. In compatible interactions amongst a set of 19 genes, more than 57·2% of differentiated transcripts from P. viticola infected-leaves (Pv-infected leaves) and 90% from E. necator-infected leaves (En-infected leaves) were down-regulated at 24 h post-inoculation (hpi), indicating a manipulation of host responses by the pathogens. BTH treatment enhanced grapevine defences, with pathogen growth inhibited by 61–98%, depending on the pathogen isolate. Treatment also triggered up-regulation of pathogenesis-related protein genes such as PR-1, PR-2, PR-3, PR-8 and PR-10 in Pv-infected leaves, and PR-3, PR-6 and PR-10 in En-infected leaves. Treatment with BTH also led to regulation of indole pathway transcripts; in particular, anthranilate synthase was down-regulated at 24 hpi in all infected leaves, then strongly up-regulated afterwards according to the rate of pathogen development. Quantitation of polyphenols and stilbenes showed that pterostilbene was specifically accumulated in pre-treated leaves and associated with biological efficacy and significant increases in PR protein gene transcripts. The temporal evolution of defence-related genes in pre-treated infected leaves suggests that grapevine responses vary depending on the inter- or intra-species variability of pathogens.