This study aimed to investigate the efficiency of the secondary metabolites (SMs) produced by a co-culture of Trichoderma harzianum TW21990 and Burkholderia vietnamiensis B418 in the control of Colletotrichum siamense CM9. A fermentation filtrate of B418 + TW21990 co-culture (BT21) produced a notable increase in the inhibition rate of CM9 compared to those of TW21990 and B418 monocultures, which reached 91.40% and 80.46% on PDA plates and strawberry leaves, respectively. The BT21 fermentation broth exhibited high control efficiency on strawberry root rot of 68.95% in a pot experiment, which was higher than that in the monocultures and fluazinam treatment. In addition, BT21 treatment promoted strawberry root development, improved antioxidative enzyme activities in the leaves and roots, and enhanced the total chlorophyll content of the strawberry leaves. UHPLC-MS/MS analysis of fermentation filtrates was performed to elucidate SM variations, revealing 478 and 795 metabolites in BT21 co-culture in positive and negative ion modes, respectively. The metabolomic profiles suggested abundant SMs with antagonistic capabilities and growth-promoting effects: 3-(propan-2-yl)-octahydropyrrolo [1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Val)), 3-[(4-hydroxyphenyl)methyl]-octahydropyrrolo[1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Tyr)), 3-indoleacetic acid (IAA), 2-hydroxycinnamic acid, 4-aminobutyric acid (GABA), bafilomycin B1, and DL-indole-3-lactic acid (ILA) were significantly enhanced in the co-culture. Overall, this study demonstrates that a co-culture strategy is efficient for inducing bioactive SMs in T. harzianum and B. vietnamiensis, which could be exploited as a novel approach for developing biocontrol consortia.