Grapevine trunk diseases (GTDs), particularly Esca, represent a major challenge for viticulture worldwide, leading to substantial economic losses. With no effective control treatments available, developing new methods such as biocontrol is crucial for managing GTDs. Our aim was to select biocontrol bacteria effective against the white-rot fungal pathogen Fomitiporia mediterranea (Fmed) and to investigate their mechanisms of action. A stepwise screening of 58 bacterial strains was conducted in vitro to assess their ability to inhibit Fmed growth through volatile and diffusible metabolites production. The screening was also done on wood sawdust from seven different grapevine cultivars. Out of 58 tested strains, 49 inhibited Fmed growth by over 50 % through their volatile organic compounds, only eight achieving this through their agar-diffusible metabolites. Pseudomonas lactis SV9, Pseudomonas paracarnis S45, and Paenibacillus polymyxa SV13 exhibited a strong efficacy in inhibiting Fmed on wood sawdust in a cultivar-dependent manner. We selected these strains for whole genome analysis and metabolomic profiling via LC-MS/MS for diffusible compounds and SPME GC-MS for volatile compounds. P. polymyxa SV13 inhibited Fmed primarily through diffusible metabolites, producing mainly fusaricidin-type compounds. Conversely, Pseudomonas strains acted mainly via their volatile metabolites, producing mainly the antifungal compound dimethyl disulfide. Genome analysis of the three bacterial strains revealed gene clusters responsible for regulating both direct and indirect mechanisms in biocontrol agents (BCAs). Our findings highlight the importance of comprehensive studies that combine in vitro experiments mimicking field conditions, with detailed investigations into modes of action to improve BCAs efficacy.