This study was performed to isolate and characterize Trichoderma species from the rhizosphere of holm oak, olive and lavender in a nursery to select effective antagonistic and growth-promoting agents and use them to amend nursery plant growth substrates. Among the isolated Trichoderma species, three from olive (T. asperellum T2, T. hamatum T3, and T. harzianum T6), three from holm oak (T. hamatum T19, T. asperellum T20, and T. virens T21) and two from lavender (T. asperellum T12 and T. harzianum T14) were selected for the in vitro antagonism test against the plant pathogens Sclerotinia sclerotiorum, Rhizoctonia solani, Verticillium dahliae, Phytophthora nicotianae and P. cinnamomi. The mycelial growth of each target species was differently and directly affected by each Trichoderma isolate and its volatile and non-volatile metabolites, suggesting multiple mechanisms in the antagonistic activity. The ability of Trichoderma isolates to trigger defense pathways in Arabidopsis was evaluated in an in vitro system by assessing the activation of the R2R3-MYB-like transcription factor gene myb72. The activation of myb72 induced by the isolates was significantly different from the control, with T. asperellum T20 as the most effective. The application of a mixture of Trichoderma isolates decreased the root rot that was caused by the artificial inoculation of R. solani and S. sclerotiorum but to a lesser extent. The highest efficacy against the oomycete P. nicotianae was shown by T. harzianum T6 alone, which produced antibiotic compounds that were effective in vitro against oomycetes. The application of a combination of local antagonists through the amendment of potting soil may have potential in the sustainable production of nursery plants.