Biofumigation refers to the suppression of soil-borne pests and pathogens by biocidal compounds released by Brassicaceous green manure and rotation crops when glucosinolates (GSLs) in their tissues are hydrolysed. We investigated the effect of environment and ontogeny on the GSL production, and thus biofumigation potential, of eight entries from five Brassica species. The environments included autumn and spring sown field plots (FA and FS) and potted plants grown under ambient conditions (PAM) or in a temperature controlled glasshouse at 20 °C/12 °C (PTC). GSL concentration was measured in the root and shoot tissue at buds-raised, flowering and maturity. Of particular interest was the suitability of the pot-grown plants for screening large numbers of brassicas for GSL production. The type of GSLs present in the tissues and their relative proportions remained relatively constant across environments and at different growth stages, with the exception of an increase in indolyl GSLs in the FS environment suspected of being induced by insect attack. Total GSL concentration generally declined from buds-raised to flowering in all environments, and was lowest at maturity. The exceptions were B. campestris, which had higher GSL concentration at flowering than at buds-raised, and the PTC environment in which most species also showed an increase at flowering. Despite GSL types and their proportions remaining relatively constant, the total GSL concentration in the root and shoot tissue of all entries varied significantly with environment (3–10-fold) and was generally ranked FS>PAM>FA>PTC. Interactions between species and environments meant that the ranking of the Brassica entries for total shoot and root GSL concentration changed with environment. However within three entries from B. napus, the ranking was consistent across the environments. The added effect of environment on phenological development and biomass production further influenced GSL production (the product of GSL concentration and biomass) on a ground area basis. The results suggest that glasshouse environments can be used to determine the types and proportions of GSLs present, and to rank entries within, but not between species for the total concentration in the tissues. However the influence of the environment on both GSL concentration and biomass production suggests that an accurate estimate of GSL production on a ground area basis to assess biofumigation potential will require measurement in the target environment.