This study describes the activity-guided isolation and purification of a novel antimicrobial protein from the seed ofCroton tigliumLinn. Purification was carried out by (NH4)2SO4 precipitation, gel filtration and DEAE-cellulose ion-exchange chromatography. Antifungal and antibacterial activities were determined after each purification step. SDS-polyacrylamide gel electrophoresis revealed that the purified protein was a monomer with molecular mass of 50 kDa. This is a first report on purification of a protein fromCroton tiglium, which possesses a strong and broad spectrum antimicrobial activity. Copyright © 2008 John Wiley & Sons, Ltd.