Cover crops, a soil conservation practice, are amongst tools that can contribute to reducing disease pressure caused by Pseudomonas syringae, considered as one of the most important bacterial plant pathogens. Indeed, it has recently been demonstrated that phyllosphere (leaf surface) bacterial community structure changed when squash (Cucurbita pepo) is grown with a rye cover crop treatment, followed by a decrease of P. syringae symptoms. Application of biocontrol agents is a known agricultural practice in integrated pest management procedures to mitigate crop losses due to microbial disease. In this study, we pursue the hypothesis that some phyllosphere bacteria promoted when squash are grown on cover crops could be isolated and used as a biocontrol agent to decrease P. syringae symptoms. We grew squash during a two-year field experiment using four agricultural practices: bare soil, cover crops, chemically terminated cover crops and plastic cover. We sampled squash leaves at 3 different dates each year and constructed a bacterial collection of cultivable strains isolated from squash leaves and rye cover crop material. Each isolated strain was identified by 16S rRNA gene sequencing and subjected to an in vitro (Petri dish) and in vivo (greenhouse) competition assay against P. syringae. Four bacterial isolates belonging to the genera Acinetobacter, Pseudomonas, Delftia and Rhizobium were proven to inhibit P. syringae growth and symptom development. Strikingly, the antagonist impact on P. syringae was stronger on older leaves. This study shed light on the importance of bacterial isolation from different sources to promote disease control