Negative environmental impacts of chemical pesticides used for pest and disease management are an increasing issue. So, safe alternatives are being developed. One of the most effective methods is to use microbial biocontrol agents as antifungal biopesticides. The aim of this study was to explore the mode of action and evaluate the potentiality of the novel bio-fungicide T34 Biocontrol ^® (Trichoderma asperellum strain T34) against strawberry fruit-rot-causing fungal pathogens. Different fungal plant pathogens were isolated from diseased fruits of strawberry showing rots symptoms, collected from a field (Royal Fruits Farm, fresh planting system) at Abo-Ghaleb Al-Mahatta, Abo-Ghaleb city region, Giza Gov., Egypt. Ten isolates were isolated and identified successfully.  Acremonium butyri, Alternaria tenuissima, Fusarium oxysporum, F. sporotrichioides, F. subglutinans, Mucor hiemalis, Rhizoctonia fragaria, R. solani and Trichothecium roseum were associated with (as causal complex of) strawberry fruit rot diseases. The commercial formulation of T34 Biocontrol ^® (Trichoderma asperellum strain, T34) was evaluated under lab conditions (in vitro) against these isolates throw two different techniques, growing on Trichoderma asperellum-mixed PDA medium plates and dual-culture. Trichoderma asperellum strain, T34 showed good antagonistic activities and significantly inhibited the fungal growth of all fungal pathogens tested. Also, the separation of chemical substances of partially purified culture filtrate of T. asperellum by GC-MS clarified that different chemical compounds with generally good antimicrobial, antibacterial, bactericidal, antifungal, antioxidant, anti-inflammatory, local anesthetic, antinociceptive, cicatrizing, antiseptic or anticancer properties can be produced by the tested biocontrol agent T34 and plays as antibiotics inhibiting the growth of plant pathogens.  Separation of chemical substances of partially purified culture filtrate of T. asperellum by GCMS, showed that T. asperellum produces compounds (e.g., 9- Octadecenoic acid, Anthrone, Thymol, Glycerol, 1-Hexadecanol,2- methyl-, D-Mannitol, Palmitic Acid, Oleic Acid, Stearic acid, 1- Monopalmitin, Acetic acid, hydrazide, Citronellene, Carveol 1, Linalool, Stanozolol and Arachidonic acid) with antimicrobial activities according to available literature. In vivo assay of the biocontrol agent T34 Biocontrol ^® under open field conditions against strawberry fruit rot diseases showed that the field application (spraying) of T34 significantly reduced the disease (fruit rots) incidence preharvest (on the field) and disease incidence and severity postharvest (during cooled storage) in both frequently tested winter seasons 2020-2021 and 2021-2022. T34 achieved highly efficient protective performance and led to a disease reduction percentage exceeding 90 % in some trials. Our results were nearly comparable with disease control achieved by comparative chemical fungicides of Fabolous 75%WP (Tebuconazole 12.5%+Chlorothalonil 62.5%(w/w)), Fabric 30%SC (Tebuconazole 15%+Kresoxim-Methyl 15%(w/v)), Fango 50%WG (Pyraclostrobin 50%(w/w)), Klop 50%WP (Difenoconazole 6%+Thiophanate methyl 44% (w/w)), Maven 18.7%WG (Pyraclostrobin 6.7%+Dimethomorph 12%(w/w)) and Mystic 20%WP (Pyrimethanil 20%(w/w)).