Background: Biocontrol of grapevine trunk diseases (GTDs) is a friendly alternative to chemical control that cause resistance and contamination of the ecosystems. Objective: isolate native grapevine root and rhizosphere microorganisms with biocontrol potential of grapevine trunk fungi diseases. Methodology: The competition for substrate (CS) was studied with the Bell's scale, contact time (CT) between the antagonist-pathogen (days) and the growth inhibition percentage (GIP). Results: Seven Trichoderma strains were isolated (from roots: Tr-1, Tr-2, Tr-3, Tr-6; and from rhizosphere: Tr-4, Tr-5 Tr-7); six Bacillus spp. (from roots: Bac-4, Bac-5, Bac-6; and from rhizosphere: Bac-1, Bac-2, Bac-3); and two Streptomyces sp. (Act-1, Act-2). The CS results evaluated with the Bell scale showed Tr-1, Tr-7, T.atv (Trichoderma atroviride) and T-22 (Trichoderma harzianum) were placed in class 1 against Lasiodiplodia theobromae and, all isolates were placed in class 1 against Campylocarpon pseudofasciculare and Phaeoacremonium parasiticum. The CT of Trichoderma was 2 days (L. theobromae) and 3 days (C. pseudofasciculare and P. parasiticum). In the antibiosis test, Tr-5 (Trichoderma) and Bac-3 (Bacillus) had the highest GIP values (>50%) against the different evaluated pathogens. The isolate Act-2 (Streptomyces sp.) presented a GIP, >70% (L. theobromae), and >40% (C. pseudofasciculare) and >30% (P. parasiticum). Implications: The use of biological control in the management of grapevine trunk diseases is an effective tool, and can be integrated into an integrated management strategy for these pathologies. Conclusions: The native isolates Bac-3 (Bacillus spp.), Tr-5 (Trichoderma spp.), Act-2 (Streptomyces sp.) demonstrated in vitro control against the pathogens L. theobromae, C. pseudofasciculare and P. parasiticum, pathogens associated to grapevine trunk diseases.