In this research, we aimed to isolate and identify native strains of Trichoderma spp. with potential activity against avocado pathogens (Neofusicoccum parvum, Colletotrichum gloeosporioides, Diaporthe sp., and Phomopsis perseae). Strains of Trichoderma spp. were isolated from roots and soil obtained from avocado orchards from different regions of Mexico. Twenty-five Trichoderma spp. strains were isolated, of which six (TSMICH7, TSMICH8, TRMICH9, TSMICH10, TSMICH15, and TRJAL25) showed greater antagonistic capacity in vitro (>80%) against avocado pathogens. After 96 h, the antagonistic strain undergoes a thickening of hyphae, while the phytopathogen tends to thin, except for Diaporthe sp., which tends to thicken. The characterization of these strains was carried out through morphological observations and the amplification and sequencing of rDNA fragments (ITS regions), as well as the translation elongation factor 1-alpha (Tef 1-α), achieving the identification of Trichoderma harzianum. However, in the in vivo evaluation (applying directly to the avocado fruit), the TSMICH7 strain maintained considerably high effectiveness (>90%) against the four phytopathogens tested, mainly with P. perseae, N. parvum, and Diaporthe sp., increasing the activity of glucanases and chitinases. Therefore, T. harzianum could be used as a biological control agent to inhibit post-harvest pathogens in avocados, thus avoiding significant losses of this fruit of international importance.