Gray mold is a serious rot in postharvest tomato fruits caused by the fungal pathogen Botrytis cinerea. Botrytis is the most prevalent postharvest fungi that cause major losses in fresh fruits, vegetables, and ornamentals as well as can infect more than 500 plant species. This investigation was designed to evaluate the effect of Trichoderma culture filtrate on the fungal pathogen in vitro and in vivo conditions. The culture filtrate significantly reduced B. cinerea growth by 94.6, and 73.7% at concentrations of 70, and 60%, respectively. Moreover, the culture filtrate of T. reesei at 70% concentration caused an enormous reduction in incidence and severity of the disease by 80.5, and 90.5% in comparison with the untreated group respectively. The secondary metabolites of several species of Trichoderma had great antifungal activity on the pathogen B. cinerea, thereby might be a promising and eco-friendly strategy for controlling gray mold in tomatoes and various postharvest fruits and vegetables.