Beneficial fungal strains of the genus Trichoderma are used as biofungicides and plant growth promoters. Trichoderma strains promote the activation of plant defense mechanisms of action, including the production of phenolic metabolites. In this work, we analyzed the effects of selected Trichoderma strains (T. asperellum KV906, T. virens GV41, and T. harzianum strains TH1, M10, and T22) and their metabolites (harzianic acid and 6-pentyl-α-pyrone) on drupes of young olive trees (4-year-old) cv. Carolea. This study used the untargeted analysis of drupe metabolome, carried out by LC–MS Q-TOF, to evaluate the phenolics profiles and target metabolomics approach to detect oleuropein and luteolin. The untargeted approach showed significant differences in the number and type of phenolic compounds in olive drupes after Trichoderma applications (by root dipping and drench soil irrigation method) compared to control. The levels of oleuropein (secoiridoid) and luteolin (flavonoid) varied according to the strain or metabolite applied, and in some cases, were less abundant in treated plants than in the control. In general, flavonoids’ levels were influenced more than secoiridoid production. The dissimilar aptitudes of the biological treatments could depend on the selective competence to cooperate with the enzymes involved in producing the secondary metabolites to defend plants by environmental stresses. Our results suggest that using selected fungi of the genus Trichoderma and their metabolites could contribute to selecting the nutraceutical properties of the olive drupe. The use of the metabolites would bring further advantages linked to the dosage in culture and storage.