Trichoderma species are well-known biological control agents. In this study, secondary metabolites from Trichoderma spp. (Trichoderma harzianum - Tr01-333257, T. viride- Tr02-333256) were extracted from liquid culture using ethyl acetate and tested for their activities against fungal pathogen Fusarium oxysporum (Fu04 - KY977424). The fungal pathogen Fusarium responsible for wilt disease observed on Solanum lycopersicum L. (Tomato) treated with Trichoderma extract. This experiment was conducted by different treatments such as Control, Tr01, Tr02, Fu04, Tr01+ Fu04, Tr02+ Fu04. Trichoderma harzianum (Tr01) showed strongest inhibitory activities against Fusarium oxysporum. The expression of two pathogenesis- related gene SlWRKY31, SlWRKY4, which are responsible for production of glucanase and chitinase contributing to pathogen inhibition. In this results showed the potential for use of Trichoderma metabolites as biological control agents against Fusarium wilt disease.