Ginkgo biloba extract (GBE) is a popular phytomedicine and has been used for disorders of the centralnervous system, cardiovascular, renal, respiratory, and circulatory diseases. Although GBE is a complexmixture of over 300 compounds, its major components are 24% flavonoids and 6% terpene lactones. In thisstudy, we tested the inhibitory effects of the three major flavonoids (kaempferol, quercetin, and isorhamnetin)from GBE, independently and as mixtures, on aromatase activity using JEG-3 cells (human placental cells)and recombinant proteins (human placental microsome). In both systems, kaempferol showed the strongestinhibitory effects among the three flavonoids; the flavanoid mixtures exerted increased inhibitory effects. Theresults of exon I.1-driven luciferase reporter gene assays supported the increased inhibitory effects of flavonoidmixtures, accompanied by suppression of estrogen biosynthesis. In the RT-PCR analysis, decreased patternsof aromatase promoter I.1 mRNA expressions were observed, which were similar to the aromatase inhibitionpatterns of flavonoids and their mixtures. The present study demonstrated that three flavonoids synergisticallyinhibit estrogen biosynthesis through aromatase inhibition, decrease CYP19 mRNA, and induce transcriptionalsuppression. Our results support the usefulness of flavonoids in adjuvant therapy for breast cancer by reducingestrogen levels with reduced adverse effects due to estrogen depletion.