The water extract of mustard seed was fractionated by solvents with an increase of polarity, and antimicrobial activities of each extracts were examined, and volatile compounds of each extract were identified by GC-MS. When the water extract was fractionated with solvents, the antimicrobial activities were high in the order of chloroform, ethylacetate, hexane, butanol and aqueous layer. In chloroform fraction, 16 volatile compounds, including 2 isothiocyanates such as 3-isothiocyanato- 1-propene and 4-isothiocyanato- 1-butane, 1 nitrile and 4 acids were identified, their contents were higher than other fractions. Twelve, 10, 4 and 7 volatile compounds were identified in ethylaceate, hexane, butanol and water fractions, respectively. The volatile compounds were considerably less in the fractions of butanol and water than others.