Isothiocyanates (ITCs) released from Brassica crops or seed meal amendments incorporated into soil have the potential to suppress pest and disease organisms in soil. We investigated in vitro toxicity of six ITCs to the mycelial growth of five cereal root pathogens (Gaeumannomyces graminis var. tritici, Rhizoctonia solani, Fusarium graminearum, Bipolaris sorokiniana, Pythium irregulare) by either adding them to the headspace above, or dissolving them in the growing media. Four alkenyl aliphatic ITCs (methyl-ITC, propenyl-ITC, butenyl-ITC, pentenyl-ITC) and two aromatic ITCs (benzyl-ITC and 2-phenylethyl-ITC) were tested. Aromatic ITCs were less toxic in the headspace experiments due to their lower volatility which reduced the headspace concentration, but were more toxic than the aliphatic ITCs when dissolved in the agar. In both experimental methods, the toxicity of the aliphatic ITCs decreased with increasing length of the side chain although there was little difference between methyl-ITC and propenyl-ITC in the headspace experiment. The fungi differed in sensitivity to the ITCs. Gaeumannomyces was the most sensitive, Rhizoctonia and Fusarium intermediate and Bipolaris and Pythium the least sensitive. Pythium was 2–16 times more resistant than the other fungi to the ITCs dissolved in agar and, in contrast to the other fungi, was more sensitive to the aliphatic ITCs than the aromatic ITCs. Suppression of some fungi by propenyl ITC and 2-phenylethyl ITC, principal products of glucosinolate hydrolysis in Brassica tissue, was superior to that of the synthetic fumigant methyl-ITC, suggesting an important role for these compounds in the pest suppression potential of brassicas. The variation in toxicity of different ITCs to the fungi (up to 7 fold) suggests there is significant scope to enhance the biofumigation potential of brassicas by selecting those which produce large quantities of GSL precursors to the most toxic ITCs for the target organism.