Trichoderma is the most studied and used fungal agent in biological disease control worldwide. Its prospection is a necessary routine, in order to select more effective and specific strains for the different existing agro pathosystems. This work reports the in vitro antagonism (Mycelial Growth Inhibition - MGI) of five Trichoderma isolates, obtained from rhizospheric and organic soil of medicinal plants cultivated in Brazil, to five different phytopathogenic fungi and their molecular identification based on actin (act), calmaldulin (cal), rDNA gene (ITS) and translation elongation factor 1-α (tef1-α). Regarding the fungus Macrophomina phaseolina, the MGI varied between 63.33 and 67.03%; for Fusarium verticillioides between 67.20 and 85.92%; Phaeocytostroma sacchari between 84.00 and 92.90%; in the case of Sclerotinia sclerotiorum, the inhibition was total (100%), and for Sclerotium rolfsii, the antagonism was between 62.03 and 79.07%. According to the molecular phylogeny performed, concatenated analysis of the genetic markers revealed that the five antagonist fungi belong to the Trichoderma afroharzianum species. It is concluded that the T. afroharzianum isolates evaluated showed good levels of in vitro control of the plant pathogenic fungi in question and will be studied via in vivo tests and in plant growth promotion.