Onion (Allium cepa) is one of the most important horticulture crops in Costa Rica and the world due to its medicinal and nutritional properties. This crop is susceptible to a wide range of diseases caused mainly by soil fungi, such as Sclerotium cepivorum, Fusarium oxysporum and Setophoma terrestris; leading farmers to recur primarily to chemical products as their main fighting strategy, which increases pollution, risk to human health, and production costs. A promising alternative to this problematic is the biological combat using antagonistic microorganisms and systemic resistance stimulators. Trichoderma spp. is a genus of filamentous fungi with worldwide distribution, which is considered a general degrader of cellulose and chitin, associated to decomposing organic matter, the rhizosphere and as a parasite to other fungi. Each isolated species of the Trichoderma genus can present different levels of specialization in the different areas mentioned. Therefore, it is essential to perform the adequate microorganism species identification, when being used as a biological control strategy. Hence, the objective of this research study was the characterization and identification of five isolates of Trichoderma spp. found in onion (A. cepa) roots, through the Scanning Electron Microscope (SEM), as well as the amplification and sequencing of the ITS1, 5.8S and ITS2 regions of the ribosomal RNA.