Aims: This study explores the biofumigation effects of glucosinolate (GSL) containing Brassica oleracea plant material on beneficial, non-target soil organisms, and aims to relate those effects to differences in GSL profiles. Methods: Leaf material of purple sprouting broccoli ‘Santee’, Savoy cabbage ‘Wintessa’, and the wild B. oleracea accession Winspit was analysed for GSL production and used for biofumigation experiments on the beneficial soil invertebrates, Folsomia candida (springtail) and Eisenia andrei (earthworm) and the soil bacterial community. Results: When mixed into soil, the Winspit plant material exerted the highest toxic effects on beneficial soil invertebrates by reducing survival and reproduction. Total GSL levels varied substantially between genotypes, in particular the aliphatic GSL (AGSL) sinigrin and gluconapin being highly abundant or exclusively present in Winspit. Differences between the genotypes regarding biofumigation effects on the soil microbial community were only observed on a temporal basis with the largest difference in bacterial community structure after 1 week. Conclusions: The high total GSL content in biofumigated soil could explain the toxicity of Winspit for soil invertebrates. These effects are likely to be the results of high AGSL levels in Winspit. The use of wild B. oleracea crops, such as Winspit, for biofumigation practices would need a proper assessment of the overall impact on soil biota before being applied on a wide scale.