Drechslera graminea is the causal agent of leaf-stripe disease in barley leading to substantial crop losses. The current work aimed to investigate the antagonistic activity of Trichoderma harzianum against D. graminea. Pathogen was isolated from the naturally infected barely leaves and molecularly identified. The identified isolate was deposited in GenBank with an accession number of OR827023. In vitro dual culture assay showed a decrease in D. graminea radial growth with a growth inhibition percentage of 77.8 %. T. harzianum culture filtrate (TCF) (30×106 spore/ ml) was efficiently applied in the greenhouse causing a significant reduction in final disease severity (FDS %) by 77 %, compared to the positive control. Moreover, application of TCF against the natural infection in the open field reduced FDS, the area under the disease progress curve (AUDPC), and the average coefficient of infection (ACI). Furthermore, TCF significantly improved yield components compared to the untreated control. To assess the plant response on a molecular basis, the protein profile was analyzed showing a wide range of variability. Five polymorphic bands were recognized; mainly 66, 48, 35, 29, and 16 kDa. The band with a molecular weight of 66 kDa had completely disappeared in the negative control, but was observed in the positive control and upon application of TCF. Meanwhile, the bands with molecular weights of 35, 29, and 16 kDa were recorded only in the positive control but were completely vanished in the negative control and upon application of TCF. Additionally, upon application of TCF under the infection stress, phenylalanine ammonia lyase (PAL) mRNA transcript accumulation had increased significantly in a way that exceeded two-fold of the non-infected control. Finally, it was concluded that T. harzianum was able to reduce leaf-stripe infection damage and promoted the barley-induced resistance in the susceptible cultivars; with the improvement of vegetative growth parameters, which may be a real prospect for leaf-stripe disease management in the future.