Botrytis cinerea and Neofusicoccum parvum are devastating fungal pathogens of grapevines. A total of 46 bacterial strains isolated from grapevines were screened for their antagonistic activity toward B. cinerea, which causes gray mold, and N. parvum, which causes cankers, trunk diseases and berry rot. In vivo screening using detached berries and leaf discs and in planta screening using growing grapevine cuttings were compared to select the most efficient biological control agents. On grapevine leaf discs, the most effective strain, Bacillus ginsengihumi, exhibited more than 80% antagonistic activity against the two major grapevine subpopulations of Botrytis cinerea (transposa and vacuma). At the berry surface, the efficacy of the bacterial strains strongly depended on the presence or absence of wounds. On stem cuttings, some strains, notably Pantoea agglomerans, were able to significantly reduce the length of the necrosis caused by N. parvum. Thus, both the plant parts and the pathogen species may significantly affect the efficacy of the bacterial strain. Marked differences between the two fungal pathogen species in terms of strain inhibitory efficacy were observed on wounded berries, as some Bacillus spp. strains efficiently controlled B. cinerea but not N. parvum. Noticeable differences in bacterial antagonist efficiencies were detected between berries and leaf discs inoculated with B. cinerea (transposa), e.g., some Paenibacillus spp. strains were efficient on leaves but not on berries. Synergistic relationships were also revealed between several bacterial strains and the pathogens. Some bacterial strains (e.g., Bacillus licheniformis) reduced B. cinerea rot severity in wounded and unwounded berries, but they increased N. parvum necrosis on stem cuttings.