Fusarium species represent a significant threat to pasture health, necessitating the development of sustainable solutions. This study explores the potential of regionally adapted Trichoderma isolates for controlling Fusarium avenaceum and promoting plant growth in the grasslands of the Iberian Peninsula. To this end, seven Trichoderma isolates (belonging to T. koningiopsis, T. koningii and T. gamsii) were obtained from soils of Extremadura and then evaluated as potential biocontrol agents against Fusarium avenaceum. For the purposes of this evaluation, water was used as a negative control, while a commercial Trichoderma product served as a positive control. An initial in vitro evaluation revealed that six Trichoderma isolates significantly inhibited F. avenaceum in a dual culture assay, reducing pathogen growth by 18 to 49%. Additionally, two of the isolates showed antifungal potential during the evaluation of their culture filtrates. Subsequently, two greenhouse assays were conducted to assess the effects of Trichoderma isolates and the pathogen on the development of Lolium perenne. One focused on seed germination and the other on established plants. The greenhouse experiments indicated that T08 (T. koningiopsis), T14 (T. koningii), and T19 (T. gamsii) significantly improved seed germination and plant growth, even outperforming the positive control on total dry matter in pathogen-infected plants during the postemergence test. Our study highlights the potential of Trichoderma isolates, particularly T08, T14, and T19, to boost plant growth and control Fusarium avenaceum in Lolium perenne. It emphasizes the importance of in planta testing and reveals the varying effects of the isolates throughout the plant cycle.