Bioassay-guided sequential extraction of the seeds of Persicaria lapathifolia followed by chromatographic separations resulted in the identification of three chalcones; flavokawain B (1), pinostrobin chalcone (2) and pashanone (3). The structures of the isolated compounds were established based on NMR and MS spectroscopic data. Disk diffusion method was employed to evaluate the antibacterial and antifungal activities of the isolated compounds against two bacterial and four fungal strains. The chloroform extract and the compounds demonstrated significant antibacterial activities. Compound 2 showed the highest activity against Staphylococcus aureus bacterial strain and Trichoderma spp fungal strain. Its antifungal activity (22 mm) against Trichoderma spp is even greater than that of the reference drug, clotrimazole (20 mm).