Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.