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This study examined the potential use of three bacterial strains—<i>Paraburkholderia</i> sp. strain CRV74, <i>Pseudomonas</i> sp. strain CRV21, and <i>Acinetobacter</i> sp. strain CRV19—as biocontrol agents of <i>Botrytis cinerea</i> in grapevine. These strains were selected for their ability to inhibit <i>B. cinerea</i> growth in vitro and used in field conditions for the control of grey mould symptoms in ‘Glera’ grapes. To this end, after inoculating these microorganisms onto plants sprayed with <i>B. cinerea</i> spores, the final yield, the physicochemical characteristics of the must, disease incidence, and the possible influence on the expression of plant-defence proteins were evaluated. Strain CRV21 resulted as being the most effective in combating grey mould (−20% of disease incidence). Although yield was not affected, significantly different values of total soluble solids content was observed. Additionally, a significant up-regulation of the genes PR-1, PR-5, β-1,3-glucanase, and class III chitinase was observed. These findings highlight the potential application of strains with anti-botrytis activity as sustainable alternatives to chemical defence for the control of this pathogen.