In many vineyards around the world, Botrytis cinerea (B. cinerea) causes one of the most serious diseases of aerial grapevine (Vitis vinifera L.) organs. The control of the disease relies mainly on the use of chemical products whose use is increasingly challenged. To develop new sustainable methods to better resist B. cinerea, beneficial bacteria were isolated from vineyard soil. Once screened based on their antimicrobial effect through an in vivo test, two bacterial strains, S3 and S6, were able to restrict the development of the pathogen and significantly reduced the Botrytis-related necrosis. The photosynthesis analysis showed that the antagonistic strains also prevent grapevines from considerable irreversible PSII photo-inhibition four days after infection with B. cinerea. The 16S rRNA gene sequences of S3 exhibited 100% similarity to Bacillus velezensis, whereas S6 had 98.5% similarity to Enterobacter cloacae. On the other hand, the in silico analysis of the whole genome of isolated strains has revealed the presence of “biocontrol-related” genes supporting their plant growth and biocontrol activities. The study concludes that those bacteria could be potentially useful as a suitable biocontrol agent in harvested grapevine.