With a reduction on available chemical treatments, there is an increased interest on biological control of grapevine trunk diseases. Few studies have investigated the impact of introducing beneficial microorganisms in rhizosphere, on the indigenous soil existent microbiome. In this study, we explored the effect of two biological control agents, Trichoderma atroviride SC1 (commercial product Vintec® from Certis Belchim, Ta SC1) and Bacillus subtilis PTA-271 (Bs PTA-271), on the grapevine rhizosphere bacterial and fungal microbiome, and on plant defense expression, using High-Throughput Amplicon Sequencing and qPCR, respectively. Additionally, we quantified both Ta SC1 and Bs PTA-271 in rhizosphere overtime using digital droplet PCR. The fungal microbiome was more affected by factors such as soil type, BCA treatment, and sampling time than bacterial microbiome. Specifically, Ta SC1 application produced negative impacts on fungal diversity, while applications of BCAs did not affect bacterial diversity. Interestingly, the survival and establishment of both BCAs showed opposite trends depending on the soil type, indicating that the physicochemical properties of soils have a role on BCA establishment. Fungal co-occurrence networks were less complex than bacterial networks, but highly impacted by Ta SC1 application. Soils treated with Ta SC1, presented more complex and stable co-occurrence networks, with a higher number of positive correlations. Induced grapevine defenses also differed according to the soil, being more affected by BCA inoculation on sandy soil.