Chestnut extracts were studied for antimicrobial activity against selected microorganisms, including plant pathogens. Chestnut extract on paper discs was applied to an agar medium to evaluate the inhibition to multiple microorganisms or the extract was added at various concentrations to a culture medium to evaluate the growth of target microorganisms. Chestnut type, tissue of plants (shell, pellicle, and leaf), extraction methods, and physical characteristics were studied to determine antimicrobial activity. Most test microorganisms were inhibited by the extracts at different effective concentrations for 50% growth inhibition (EC50). Pseudomonas fluorescens was the most sensitive (EC50 = 4.4 μg/μl), Phytophthora cambivora was one of the least inhibited (EC50 = 185 μg/μl), and Cryphonectria parasitica was not inhibited. Extracts of the Japanese × European chestnut (Castanea crenata × C. sativa) 'Colossal' showed a greater inhibition than those of wild trees of the Chinese species (C. mollissima). High temperature did not affect the inhibitory effect. Extracts from chestnut pellicle had the highest concentration of antimicrobial compound, compared with leaf and shell. The active fraction contained several substances with molecular masses consistent with one flavonol glycoside and several terpenoid substances. Pellicle and shell tissue reduced radish scab disease caused by Streptomyces scabies in the greenhouse.