Biofumigation refers to the allelopathic effects of brassicas on non-desirable soil organisms in a rotation cropping system. These effects are additional to non-hosting of root diseases, responsible for much of the break-crop effect observed in a following cereal crop. We investigated the biofumigation impacts of canola on take-all disease and rhizosphere microorganisms of following wheat crops. The biofumigation potential of the canola was tested by comparing effects of Brassica and non-Brassica break-crops and by using canola varieties with differing levels of root glucosinolates, the precursors of isothiocyanates, which are thought to be the primary biofumigant molecules released by brassicas. There was no evidence that biofumigation was related to superior growth of wheat following brassicas compared with non-Brassica break-crops, nor among the wheat crops following canola varieties with differing root glucosinolate levels. However, the break-crop effect was evident in agronomic, take-all, and some rhizosphere microbial measurements of the wheat–wheat rotations compared with fallow–wheat or break-crop–wheat rotations. Thus the break-crop effect was the primary advantage conferred by the brassicas. Any additional biofumigation effect on yield or disease was not significant in our study and does not appear to be influenced by root glucosinolate content. For broad-acre cropping with current canola or mustard varieties, and adequate grass weed control, the agronomic characteristics of canola varieties chosen should take precedence over their biofumigation potential.