Isothiocyanates (ITCs) hydrolyzed from glucosinolates (GSLs) in Brassicaceae tissue are toxic to soil organisms. In this study, the effect of aliphatic and aromatic ITCs from hydrated dry Brassicaceae shoot tissues on the mycelium and oospores of the pea root rot pathogen Aphanomyces euteiches was investigated. The profile and concentrations of GSLs in two test Brassicaceae species, Sinapis alba and Brassica juncea, and the ITCs from the dominant hydrolyzed parent GSLs were monitored. The concentrations of dominant ITCs and pathogen exposure time were evaluated in in vitro experiments. The greatest effect on the pathogen was observed from aliphatic ITCs hydrolyzed from B. juncea tissue, and the effect depended on the ITC concentration and exposure time. ITCs were more effectively hydrolyzed from B. juncea GSLs than from S. alba GSLs; i.e., the ITC/GSL ratio was higher in B. juncea than in S. alba tissue, giving a different release pattern. The release of phenylethyl isothiocyanate, which was common to both species, followed a pattern similar to that of the dominant ITC in each crop species. This suggests that traits other than GSL content, e.g., plant cell structure, may affect the release of ITCs and should therefore influence the choice of species used for biofumigation purposes.