The aim of this study was to examine the efficacy of glyceollins isolated from soybean seeds elicited with Aspergillus sojae for its antifungal potential. Glyceollins I, II, and III were determined by HPLC-MS analysis. The glyceollins (200 and 600 microg/disk) revealed remarkable antifungal effect against Fusarium oxysporum , Phytophthora capsici, Sclerotinia sclerotiorum, and Botrytis cinerea, within the growth inhibition range of 10.9-61.0%, along with their respective MIC values ranging from 25 to 750 microg/mL. The glyceollins also had a strong detrimental effect on spore germination of all tested plant pathogens along with concentration- as well as time-dependent kinetic inhibition of P. capsici. Thus, the results obtained in this study demonstrate that glyceollins derived from soybean seeds elicited with A. sojae possess a wide range of fungicidal activity and could become an alternative to synthetic fungicides for controlling certain important fungal diseases.