Botrytis cinerea is a common pathogenic agent, causing a significant reduction in tomato quality and yield. In this study, the potential of two biocontrol fungal agents, Trichoderma harzianum and Clonostachys rosea to prevent and alleviate B. cinerea infection was assessed. To this end, the fungicidal effects of both biocontrol fungi were evaluated on ten common phytopathogens, including B. cinerea. Then, various ratios of T. harzianum and C. rosea mixtures were assessed to find the ratio that best promotes the growth of tomato seedlings and prevents the establishment of B. cinerea. The results showed that T. harzianum and C. rosea had significant fungicidal effects on all tested phytopathogens. Also, both biocontrol agents reduced the establishment of B. cinerea, in vitro assays, indicating they had significant fungicidal effects against this phytopathogen. However, when applied as a biofungicide to tomato plants, a combination of both biocontrol agents gave better control of B. cinerea. Investigating the molecular basis of resistance to B. cinerea induced by the mixture of T. harzianum and C. rosea revealed that the salicylic acid signaling pathway plays an important role in modulating the antioxidant enzyme activity. Taken together, the results of this research confirmed that both biocontrol fungal agents could induce the mechanism of B. cinerea resistance in tomato plants and shed light on the regulatory pathways associated with the resistance mechanism.