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BACKGROUND: Botryosphaeria dieback is a canker disease caused by fungal species of the Botryosphaeriaceae family that threatens almond productivity. The most common control measure to prevent canker development is the application of fungicides which are being phased out by European Union regulations. In the present study, two sets of bacterial strains were evaluated for their antifungal activity against pathogenic Botryosphaeriaceae species through in vitro and in vivo antagonism assays. RESULTS: The rhizospheric bacteria Pseudomonas aeruginosa AC17 and Bacillus velezensis ACH16, as well as the endophytic bacteria Bacillus mobilis Sol 1–2, respectively inhibited 87, 95, and 63% of the mycelial growth of Neofusicoccum parvum, Botryosphaeria dothidea, Diplodia seriata, and Macrophomina phaseolina. Additionally, they significantly reduced the length of lesions caused by N. parvum and B. dothidea in artificially inoculated detached almond twigs. All these bacterial strains produce hydrolytic enzymes that are able to degrade the fungal cell wall. P. aeruginosa AC17 also produces toxic volatile compounds, such as hydrogen cyanide. This strain was the most effective in controlling Botryosphaeria dieback in planta under controlled conditions at a level similar to the biocontrol agent Trichoderma atroviride and standard chemical fungicide treatments. CONCLUSION: Pseudomonas aeruginosa AC17 is the best candidate to be considered as a potential biocontrol agent against Botryosphaeriaceae fungi affecting almond. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]