AbstractTrichodermaharzianum is one of the most commonly used fungal species used in the biological control of plant pathogenic fungi. Its ability to control biological pathogens is strengthened by the production of analytic enzymes. In the identification of these fungal species, it has been noted that morphological characteristics alone are insufficient. Hence, the aim of this study was to identify 12 Trichoderma isolates based on their molecular markers and to evaluate the antagonistic activity of these Trichoderma isolates against several plant pathogens. The 12 Trichoderma/Hypocrea isolates were harvested from the rhizosphere of healthy tomato plants in Abha region, Saudi Arabia. These isolates were identified by both morphological and molecular techniques, such as the sequencing of the 5.8S-ITS region. Furthermore, based on the taxonomy and phylogeny of these isolates, the genetic similarities based on the inter-simple sequence repeat-PCR markers separated the 12 Trichoderma isolates into 2 major clusters. The 12 isolates were then used in confrontational assays with Rhizoctonia solani, Pythium ultimum, and Alternaria solani. Most of the Trichoderma isolates showed a strong antagonistic activity, which could also be observed with the use of SEM. This study highlights the possibility of using T. harzianum in IPM programs as an effective biological agent against several pathogenic fungi.