Botryosphaeria dieback, a significant grapevine trunk disease (GTD) caused by various pathogens, represents a serious threat to viticulture. Biocontrol emerges as a promising sustainable alternative to chemical control, aligning toward environmentally friendly viticultural practices. This study evaluated the in vitro, in vivo, and in situ biocontrol potential of Chilean native bacteria isolated from wild flora and endophytic communities of grapevine against Neofusicoccum parvum. In vitro biocontrol assays screened 15 bacterial strains at 10, 22, and 30 degrees C, identifying four Pseudomonas strains with >30% mycelial growth inhibition. In diffusible agar and double plate assays, plant growth-promoting bacteria AMCR2b and GcR15a, which were isolated from native flora, achieved significant inhibition of N. parvum growth, with reductions of up to similar to 50% (diffusible agar) and up to similar to 46% (double plate). In vivo experiments on grapevine cuttings revealed that strains AMCR2b and GcR15a inhibited mycelial growth (17-90%); younger grapevines (1-5 years) were more susceptible to N. parvum. In situ trials using Vitis vinifera L. cv. Cabernet Sauvignon and Sauvignon Blanc demonstrated higher fungal susceptibility in Sauvignon Blanc. These results highlight the potential of Pseudomonas sp. AMCR2b and GcR15a to be effective biocontrol agents against GTDs at a wide range of temperatures, contributing to sustainable viticulture.