Antimicrobial activity of various extracts of Xanthium strumarium L. was tested against 25 strains of bacteria, yeast and fungus. The crude ethylacetate extract exhibited strong growth inhibition to the tested strains with the exception of partial Gram-negative bacteria. The property of antimicrobial compound was very stable under heat treatment at $120^{circ}C$, but it was unstable in acid (pH 3.0) and alkali (pH 10.0) treatment. The antimicrobial compounds were purified by boiling water extraction, ethylacetate extraction, charcoal column chromatography, silica gel column chro- matography and reverse phase HPLC. The purified compound A and B were detected in a single peak (each above 98% purity) through the HPLC analysis. The compound A and B showed a strong growth inhibition against Gram-negative and positive bacteria in the agar diffusion method. When tested by the FDA method using the esterase, compound A mainly inhibited the growth of bacteria and compound B showed the growth inhibition of both bacteria and yeasts.