Priming plants with beneficial microbes can establish rapid and robust resistance against numerous pathogens. Here, compelling evidence is provided that the treatment of rapeseed plants withTrichoderma harzianumOMG16 andBacillus velezensisFZB42 induces defence activation againstVerticillium longisporuminfection. The relative expressions of the JA biosynthesis genesLOX2andOPR3, the ET biosynthesis genesACS2andACO4and the SA biosynthesis and signalling genesICS1andPR1were analysed separately in leaf, stem and root tissues using qRT-PCR. To successfully colonize rapeseed roots, theV. longisporumstrain 43 pathogen suppressed the biosynthesis of JA, ET and SA hormones in non-primed plants. Priming led to fast and strong systemic responses of JA, ET and SA biosynthesis and signalling gene expression in each leaf, stem and root tissue. Moreover, the quantification of plant hormones via UHPLC-MS analysis revealed a 1.7- and 2.6-fold increase in endogenous JA and SA in shoots of primed plants, respectively. In roots, endogenous JA and SA levels increased up to 3.9- and 2.3-fold in Vl43-infected primed plants compared to non-primed plants, respectively. Taken together, these data indicate that microbial priming stimulates rapeseed defence responses againstVerticilliuminfection and presumably transduces defence signals from the root to the upper parts of the plant via phytohormone signalling.