Beneficial microorganisms, including members of theTrichodermagenus, are known for their ability to promote plant growth and disease resistance, as well as being alternatives to synthetic inputs in agriculture. In this study, 111Trichodermastrains were isolated from the rhizospheric soil of Florence Aurore, an ancient wheat variety that was cultivated in an organic farming system in Tunisia. A preliminary ITS analysis allowed us to cluster these 111 isolates into three main groups,T. harzianum(74 isolates),T. lixii(16 isolates) andT.sp. (21 isolates), represented by six different species. Their multi-locus analysis (tef1, translation elongation factor 1; rpb2, RNA polymerase B) identified threeT. afroharzianum, oneT. lixii, oneT. atrobrunneumand oneT. lentinulaespecies. These six new strains were selected to determine their suitability as plant growth promoters (PGP) and biocontrol agents (BCA) against Fusarium seedling blight disease (FSB) in wheat caused byFusarium culmorum. All of the strains exhibited PGP abilities correlated to ammonia and indole-like compound production. In terms of biocontrol activity, all of the strains inhibited the development ofF. culmorumin vitro, which is linked to the production of lytic enzymes, as well as diffusible and volatile organic compounds. An in planta assay was carried out on the seeds of a Tunisian modern wheat variety (Khiar) by coating them withTrichoderma. A significant increase in biomass was observed, which is associated with increased chlorophyll and nitrogen. An FSB bioprotective effect was confirmed for all strains (withTh01being the most effective) by suppressing morbid symptoms in germinated seeds and seedlings, as well as by limitingF. culmorumaggressiveness on overall plant growth. Plant transcriptome analysis revealed that the isolates triggered several SA- and JA-dependent defense-encoding genes involved inF. culmorumresistance in the roots and leaves of three-week-old seedlings. This finding makes these strains very promising in promoting growth and controlling FSB disease in modern wheat varieties.