Fusarium Tri4 encodes a key cytochrome P450 monooxygenase for hydroxylation of trichodiene early in the biosynthesis of trichothecenes. In this study, we established a system for screening for inhibitors of trichothecene biosynthesis using transgenic Saccharomyces cerevisiae expressing Tri4. For easy evaluation of the TRI4 activity, trichodiene-11-one was used as a substrate and the formation of 2 alpha-hydroxytrichodiene-11-one was monitored by HPLC. Using this system, TRI4 proved to be inhibited by various flavones and furanocoumarins. We also found that a catechin-containing commercial beverage product, Catechin Supplement 300 (CS300), inhibited TRI4 activity, at a concentration which did not significantly affect the growth of the transgenic yeast. At an early stage of culture, both flavone and CS300 exhibited a toxin-inhibitory activity against Fusarium graminearum. However, inhibition of trichothecene production was not observed with longer incubation periods at minimum concentrations necessary to inhibit > 50% of the TRI4 activity, presumably due to the metabolism by the fungus. The results suggest that this yeast screening system with TRI4 is useful for the rapid identification of lead compounds for the design of trichothecene biosynthesis inhibitors that are resistant to modification by the fungus.