As critical biocontrol agents, Trichoderma and Metarhizium have demonstrated vast potential in disease prevention and pest control respectively. Current applications, however, are predominantly confined to singular use or simplistic combinations of the two fungi. This study innovatively utilizes Metarhizium and Trichoderma as experimental subjects, employing non-targeted metabolomics to compare the differences in metabolites between co-culture and separate fermentation. The metabolite distinctions in co-culture are meticulously analyzed using sample correlation, least squares discriminant analysis, cluster analysis, and KEGG enrichment analysis. Metabolome sequencing analysis is then applied to compare the co-culture fermentation filtrate with Trichoderma atroviride and Metarhizium anisopliae. Differential metabolite analysis found that co-culture had significantly up-regulated or down-regulated metabolites compared to singular cultures.However,significant metabolite discrepancies are discovered in singular cultures, notably the up-regulated metabolites such as osthole and spirocyclic amine, which have documented antibacterial functions that may prove effective against Fusarium graminearum. Ultimately, the findings from this research contribute to the exploration of synergistic applications of biocontrol fungi and serve as a valuable reference for future development of bio-pesticides for disease and pest control.