Pythium damping-off adversely affects tomato cultivation by inducing seed and seedling deterioration, markedly diminishing output. Although traditional fungicides are effective, their excessive use presents environmental and health hazards and may result in pesticide resistance. This has prompted the pursuit of sustainable damping-off management solutions that reduce dependence on chemical pesticides. Trichoderma species are environmentally sustainable and potential biological control agents. Consequently, we identified 50 Trichoderma isolates and evaluated them for their antifungal efficacy against Pythium species, the pathogen responsible for damping-off disease in tomatoes. The isolates exhibiting the most substantial inhibitory zones against Pythium species were MR11, MR19, MR25, MR33, and MR41, which were identified using MALDI-TOF as Trichoderma viride, T. asperellum, T. hamatum, T. koningii, T. longibrachiatum, and T. polysporum, respectively. The antifungal efficacy of the five chosen isolates was evaluated against phytopathogenic fungi, including Aspergillus niger, Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Helminthosporium solani, Penicillium chrysogenum, Rhizoctonia solani, Pythium aphanidermatum, and Podosphaera xanthii. T. longibrachiatum MR33 exhibited the most significant inhibition zone widths, ranging from 25 to 42, against the examined pathogenic fungi. The antifungal mechanism of T. longibrachiatum MR33 against P. aphanidermatum was investigated using scanning electron microscopy (SEM). The five chosen Trichoderma strains, MR11, MR19, MR25, MR33, and MR41, were evaluated for their secretion of several growth-promoting chemicals; MR33 exhibited a significant secretion of IAA, which enhanced tomato seed germination by 35%. In vitro experiments shown that MR33 dramatically diminished the DSS induced by P. aphanidermatum in tomatoes by 91%, while augmenting fruit weight by 45% and increasing tomato output by 60% in MR33-treated plants. Soil soaking with the MR33 strain successfully reduces APX and CAT levels to those of the unstressed control. In comparison to the infected control, the chlorophyll content in MR33-treated plants rose by 225%, whilst the carotenoid content increased by 28%. The T. longibrachiatum MR33 strain is suggested as a potential biocontrol agent for tomato dumping-off disease.