The growth-inhibiting activities of Sinapis alba L. seed-derived materials were examined on the growth of Bifidobacterium bifidum, B. breve, B. longum, Clostridium difficile, C. perfringens, Escherichia coli, Lactobacillus acidophilus, and L. casei. The active component of S. alba seeds was purified using silica gel column chromatography and HPLC and was identified as phenethyl isothiocyanate by various spectroscopic analyses. The antimicrobial activity of phenethyl isothiocyanate varied according to the dose and bacterial strain tested. Phenethyl isothiocyanate strongly inhibited the growth of C. difficile and C. perfringens at 1 mg/disc, and weakly (+) inhibited its growth at 0.1 mg/disc. Furthermore, phenethyl isothiocyanate moderately (++) inhibited the growth of E. coli at a dose of 2 mg/disc, but did not inhibit the growth of bifidobacteria and lactobacilli. Addition of various functional groups to isothiocyanates resulted in selective inhibitory activity against harmful bacteria with low concentrations of aromatic isothiocyanates demonstrating greater inhibitory activity against clostridia and E. coli than aliphatic isothiocyanates. In conclusion, aromatic isothiocyanates containing phenethyl-, benzyl-, and benzoyl-groups might be useful in the development of novel preventive and therapeutic agents against diseases caused by harmful intestinal bacteria.