Fungi in the genus Trichoderma are widespread in the environment, mainly in soils. They are used in agriculture because of their mycoparasitic potential; Trichoderma have the ability to increase plant health and provide protection against phytopathogens, making them desirable plant symbionts. We isolated, identified, and characterized Trichoderma from different regions of Saudi Arabia and evaluated the ability of Trichoderma to promote plant growth. Morphological and molecular characterization, along with phylogenetic studies, were utilized to differentiate between Trichoderma species isolated from soil samples in the Abha and Riyadh regions, Saudi Arabia. Then, plant growth-promoting traits of the isolated Trichoderma species were assessed. Eight Trichoderma isolates were characterized via morphological and molecular analysis; six (Trichoderma koningiopsis, Trichoderma lixii, Trichoderma koningii, Trichoderma harzianum, Trichoderma brevicompactum, and Trichoderma velutinum) were from Abha and two (T. lixii and T. harzianum) were from Riyadh. The isolated Trichoderma strains belonged to three different clades (Clade 1: Harzianum, Clade 2: Brevicompactum, and Clade 3: Viride). The Trichoderma isolates varied in plant growth-promoting traits. Seeds treated with most isolates exhibited a high percentage of germination, except seeds treated with the T3-T. koningii isolate. 100% germination was reported for seeds treated with the T4-T. harzianum and T6-T. brevicompactum isolates, while seeds treated with the T1-T. koniniopsis and T5-T. lixii isolates showed 91.1% and 90.9% germination, respectively. Seeds treated with the T8-T. velutinum, T2-T. lixii, and T7-T. harzianum isolates had germination rates of 84.1%, 82.2%, and 72.7%, respectively. The Trichoderma isolate T5-T. lixii stimulated tomato plant growth the most, followed by T7-T. harzianum, T8-T. velutinum, T4-T. harzianum, T1-T. koniniopsis, T2-T. lixii, and T6-T. brevicompactum; the least effective was T3-T. koningii. A maximum fresh weight of 669.33mg was observed for the T5-T. lixii-treated plants. The Abha region had a higher diversity of Trichoderma species than the Riyadh region, and most isolated Trichoderma spp. promoted tomato growth.