To investigate the antimicrobial activities of mustard seed(Brassica juncea), a series of solbents were examined for extraction of antimicrobial substances from mustard seed and then antimicrobial substances were identified by simultaneous distillation extraction(SDE) and GC-MS methods. Water and methanol extracts of mustard seed showed antimicrobial activitries against experimental strains while those from hexane extract almost was not observed. The distilled components of mustard seed by SDE method showed strong antimicrobial activities, methanol extract of residues fraction exhibited a little, and water layer with the exception of distilled components showed no antimicrobiol activities. The 30 varieties of distilled components including 3 types of isothiocyanate such as 3-isothiocyanate-1-propene, 4-isothiocyanate-1-butene, and 3-isothiocyanatoethyl benzene were identified from mustard seed.