This study examined the potential use of three bacterial strains—Paraburkholderia sp. strain CRV74, Pseudomonas sp. strain CRV21, and Acinetobacter sp. strain CRV19—as biocontrol agents of Botrytis cinerea in grapevine. These strains were selected for their ability to inhibit B. cinerea 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 B. cinerea 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.