Endosymbiosis is very common between bacteria and insects, and it has been deeply studied for over a century on model insects such as Bactrocera oleae, the key pest of the olives. It was demonstrated that “Candidatus Erwinia dacicola” is the main component of its midgut bacterial communities, acting a fundamental role in the fly’s nutrition process and thus on its fitness. In this study, Trichoderma secondary metabolites have been used to treat olive fruit fly in order to alter the “Ca. Erwinia dacicola” titer and to assess the subsequent effects on its host. The selected metabolites, 6-pentyl-α-pyrone and harzianic acid, directly affect the insect’s fitness also on the subsequent generation, but not always in a concentration-dependent manner. Aside from the direct effects, the treatments also showed a modification of the bacterial titer. Therefore, real-time qPCRs were carried out on wild individual flies highlighting natural variations of the symbiont presence and activity during the seasons. The data obtained suggest that bioactive fungal metabolites can be formulated for direct or indirect control strategies of B. oleae in integrated pest management programs.