Bioassay-guided fractionation of the crude extract of the whole plant of Macleaya cordata R. Br. led to the isolation of four alkaloids, which were identified as sanguinarine (1), chelerythrine (2), protopine (3) and alpha-allocryptopine (4) on the basis of their physicochemical and spectrometric data. Compounds 1 and 2 demonstrated a significant antifungal activity against the six test fungi with median inhibitory concentrations (IC50) ranging from 0.47 to 6.13 microg/mL. Compound 1 was the most effective with an IC50 of 0.47 microg/mL on Rhizoctonia solani. Furthermore, compounds 1 and 2 also demonstrated strong antibacterial activity, with IC50 values ranging from 5.01 to 11.3 microg/mL, and minimum inhibitory concentrations (MIC) ranging from 8.0 to 32.0 microg/mL. This is the first report on the activity of the alkaloids from M. cordata against economically important phytopathogenic fungi and bacteria.