The genus Trichoderma plays a vital role in agriculture by promoting plant growth, enhancing nutrient uptake, and protecting crops from pathogens through biocontrol mechanisms. This can be largely attributed to its production of diverse secondary metabolites (SMs), including epidithiodiketopiperazines (ETPs). Our previous study has reported the complex biosynthesis of α, β’-disulfide bridged ETPs, in which TdaH and TdaG are highly conserved in catalyzing C6’-O-methylation and C4, C5-epoxidation, respectively. Here we proved the functional diversification of ETP methylation and oxidation by TdaH and TdaG towards eleven pathogenic fungi, including Fusarium, Aspergillus, and Botrytis species. Elimination of C6’-O-methylation and C4, C5-epoxidation reduced the antagonistic effects of Trichoderma hypoxylon against various pathogenic fungi. However, each deletion mutant showed varying antagonistic effects against different pathogenic fungi. Our results highlight the importance of ETP structural diversity in T. hypoxylon‘s ecological adaptation and biocontrol potential, offering insights into developing enhanced antifungal agents against plant pathogens.