Trichoderma has been used as an eco-friendly biocontrol agent for different crop species. Not only does it enhance plant resistance, but it also improves plant growth. Therefore, in this study, Trichoderma viride and Trichoderma harzianum were used as protective and curative treatments to trigger the systemic defense of tomato plants against tomato mosaic virus (ToMV). T. viride isolate Tvd44 and T. harzianum isolate ThDA66 were identified and molecularly characterized before being used as antiviral agents against ToMV. The HPLC analysis results of Tvd44 and ThDA66 culture filtrates revealed that the most detected active biomolecules had a range of biological activities, including antiviral capabilities. The foliar spraying application of Tvd44 or ThDA66 filtrates against ToMV-inoculated tomato plants significantly improved plant growth parameters. Different Trichoderma treatments improved tomato plants' chlorophyll content, phenolics, flavonoids, and DPPH. Additionally, the antioxidant enzymatic activities of tomato plants that were ToMV-inoculated and non-inoculated were enhanced compared to those of untreated plants. Moreover, different Trichoderma treatments exhibited a decrease in the oxidative stress markers in all treatments compared to plants that were inoculated with ToMV alone. The q-PCR analysis of genes from different pathways, including PAL-1, CHS, POD, PR-2, JERF3, and WRKY19, showed significant gene expression in all Trichoderma treatments, showing systemic resistance. In conclusion, the present investigation proved the effective role of Trichoderma as a biocontrol agent against ToMV in tomato plants.