Chestnuts, a valuable tree in Europe, the US, and Asia, are cultivated for their culinary and therapeutic properties. However, chestnut blight disease, a major biotic agent, has led to the death of almost 50% of orchard trees since its introduction in 1960. Molecular identification of C. parasitica (the causal agent of chestnut blight) has been done in this research with ITS and TEF primers. The use of antagonistic microbes for biological control may be an effective solution for chestnut blight canker, in this case, eight different Trichoderma species were tested against virulent and hypovirulent samples of C. parasitica, with six showing good biocontrol and inhibition against the pathogens. This study highlights the effectiveness of antagonist fungi in treating chestnut blight canker.