This study analyzes the in vitro antagonistic activity of Trichoderma sp. isolates against Fusarium subglutinans and evaluates the effect of the pine seeds treatment with Trichoderma sp. on the incidence of root rot. Twelve Trichoderma sp. isolates and two F. subglutinans isolates were included in the study. Trichoderma sp. inhibited F. subglutinans mycelial growth through direct contact with hyphae and the production of volatile antifungal compounds. Pine seeds treatment with the antagonist Trichoderma sp. reduced the incidence of root rot, increased the emergence and initial growth in the height of seedlings, and improved seedling health.