| Summary: | Grapevine trunk diseases (GTDs) are caused by multiple unrelated fungal pathogens, and their management remains difficult worldwide. Biocontrol is an attractive and sustainable strategy given the current need for a cleaner viticulture. In this study, twenty commercial vineyards were sampled across California to isolate endophytic and rhizospheric bacteria from different grapevine cultivars with the presence and absence of GTD symptoms. A collection of 1344 bacterial isolates were challenged in vitro against <i>Neofusicoccum parvum</i> and <i>Diplodia seriata</i>, from which a subset of 172 isolates exerted inhibition levels of mycelial growth over 40%. Bacterial isolates were identified as <i>Bacillus velezensis</i> (<i>n =</i> 154), <i>Pseudomonas</i> spp. (<i>n =</i> 12), <i>Serratia plymuthica</i> (<i>n =</i> 2) and others that were later excluded (<i>n =</i> 4). Representative isolates of <i>B. velezensis</i>, <i>P. chlororaphis,</i> and <i>S. plymuthica</i> were challenged against six other fungal pathogens responsible for GTDs. Mycelial inhibition levels were consistent across bacterial species, being slightly higher against slow-growing fungi than against Botryosphaeriaceae. Moreover, agar-diffusible metabolites of <i>B. velezensis</i> strongly inhibited the growth of <i>N. parvum</i> and <i>Eutypa lata</i>, at 1, 15, and 30% <i>v</i>/<i>v</i>. The agar-diffusible metabolites of <i>P. chlororaphis</i> and <i>S. plymuthica</i>, however, caused lower inhibition levels against both pathogens, but their volatile organic compounds showed antifungal activity against both pathogens. These results suggest that <i>B. velezensis</i>, <i>P. chlororaphis</i> and <i>S. plymuthica</i> constitute potential biocontrol agents (BCAs) against GTDs and their application in field conditions should be further evaluated.
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