The Trichoderma genus, despite being applied for decades as a biological control in agriculture, has only recently been gaining a share in the biopesticide market, thanks to its versatility in controlling diseases and acting as a promoter of growth. Most Trichoderma commercial products have conidia as active ingredient, but new papers report the production of another propagule, more resistant to formulation and to application conditions in the field - the microsclerotia (MS). Thus, our objective was to evaluate the nutritional conditions that favored the greater production of MS, using two cultures of T. asperellum with known antagonistic activity. For this, we used the fractional factorial design tool, evaluating 5 variables: Carbon source, Carbon concentration, C: N ratio, Culture, Nitrogen source, on the production of MS. The results indicated that all variables were statistically significant, influencing on MS production. In the best condition tested, we obtained values higher than 104 MS mL-1, corroborate with other authors.