Fusarium root rot is a major cryptogamic disease in olive trees caused by the soil-borne fungus Fusarium solani. Controlling this disease requires the extensive use of chemicals. However, using BCAs such as some Trichoderma strains may be an opportune alternative to fungicides in protecting olive plantations. A new isolate (Fso14) was isolated from young olive trees showing severe dieback symptoms. The objective of this work was to analyze the biocontrol behavior of a Tunisian strain of T. harzianum (Ths97) on olive trees against Fso14 by assessing both mycoparasitic activity (in planta and in vitro) and ability to locally modulate different gene-related defenses of the plant. Ths97 was found to inhibit Fso14 growth in vitro. Optical microscopic analysis at the confrontation zone between hyphae showed that Ths97 grew alongside Fso14 with numerous contact points suggesting parasitic activity. On olive trees, Ths97 developed a strong protective role against root infestation by Fso14, whether inoculated before or after the pathogenic agent. When inoculated alone, Fso14 and Ths97 did not modulate (or only slightly with inhibitions or inductions, respectively) the expression of genes involved in plant immunity (oxidative stress, phenylpropanoid pathway, PR-proteins and JA/Et-SA hormonal status). However, when Ths97 was inoculated in combination with Fso14, several defense-related genes were highly up-regulated, indicating probable primed-plant events. These promising results provided valuable information on using Ths97 as a beneficial agent to control fusarium root rot disease caused by F. solani in olive trees.