One of the major and yet unsolved threats for viticulture is the group of vascular fungal infections, the so-called grapevine trunk diseases. Besides their latent nature and the enormous number of associated pathogens, their control is also hampered by the lack of effective fungicides, directing growing attention toward the use of biocontrol agents. In the present study the isolation, identification, and characterization of a bacterial strain are presented, showing biocontrol potential against some main causal agents of grapevine trunk diseases. The strain was isolated from the wood of an asymptomatic grapevine and selected for the fungicidal activity against the pathogen Phaeomoniella chlamydospora. According to 16S rDNA, gyrA, and gyrB sequences, the isolate belongs to Bacillus velezensis species. Confrontation tests with the bacterium or with its fermentation broth further revealed growth inhibition and fungicide activity against Botryosphaeria dothidea, Eutypa lata and Diaporthe ampelina pathogens. Fractionation of the bacterial culture filtrate suggests that the antifungal agents secreted by the B. velezenzis isolate are mainly lipoproteins. Phytotoxicity tests were also carried out with the isolate, showing no harmful effects on grapevine foliar disks.