Kiwifruit (Actinidia spp.) is an economically important crop and a bacterial canker disease, caused by Pseudomonas syringae pv. actinidiae (Psa), is the most destructive disease in kiwifruit production. Therefore, prevent and control of the disease is a critical issue in kiwifruit industry worldwide. Unfortunately, there is no reliable control methods have been developed. Recently, interest in disease control using microbial agents is growing. However, kiwifruit microbiota and their roles in the disease control is mainly remaining unknown. In this study, we secured bacterial libraries from kiwifruit ecospheres (rhizosphere, endospere, and phyllosphere) and screened reliable biocontrol strains against Psa. As the results, Streptomyces racemochromogenes W1SF4, Streptomyces sp. W3SF9 and S. parvulus KPB2 were selected as anti-Psa agents from the libraries. The strains showed forcible antibacterial activity as well as exceptional colonization ability on rhizosphere or phyllosphere of kiwifruit. Genome analyses of the strains suggested that the strains may produce several anti-Psa secondary metabolites. Our results will contribute to develop biocontrol strains against the kiwifruit canker pathogen and the disease management strategies.