The soil-borne fungal pathogen Verticillium longisporum is able to penetrate the root of a number of plant species and spread systemically via the xylem. Fumigation of Verticillium contaminated soil with Brassica green manure is used as an environmentally friendly method for crop protection. Here we present a study focused on the potential role of glucosinolates and their breakdown products of the model plant Arabidopsis thaliana in suppressing growth of V. longisporum. For this purpose we analysed the glucosinolate composition of the leaves and roots of a set of 19 key accessions of A. thaliana. The effect of volatile glucosinolate hydrolysis products on the in vitro growth of the pathogen was tested by exposing the fungus to hydrated lyophilized plant tissue. Volatiles released from leaf tissue were more effective than from root tissue in suppressing mycelial growth of V. longisporum. The accessions varied in their efficacy, with the most effective suppressing mycelial growth by 90%. An analysis of glucosinolate profiles and their enzymatic degradation products revealed a correlation between fungal growth inhibition and the concentration of alkenyl glucosinolates, particularly 2-propenyl (2Prop) glucosinolate, respectively its hydrolysis products. Exposure of the fungus to purified 2Prop glucosinolate revealed that its suppressive activity was correlated with its concentration. Spiking of 2Prop glucosinolate to leaf material of one of the least effective A. thaliana accessions led to fungal growth suppression. It is suggested that much of the inhibitory effect observed for the tested accessions can be explained by the accumulation of 2Prop glucosinolate.