We investigated whether ectomycorrhizal (ECM) fungal species exhibit antibacterial activity towards culturable bacterial communities in mycorrhizospheres. Four hundred and thirty bacterial strains were isolated from the ECM root tips of Pinus densiflora and bulk soil, and 21 were co-cultured with six ECM fungal species. Three hundred and twenty-nine bacterial 16S rDNA sequences were identified in ECM roots (n=185) and bulk soil (n=144). Mycorrhizosphere isolates were dominated by Gram-negative Proteobacteria from 16 genera, including Burkholderia, Collimonas, Paraburkholderia, and Rhizobium. Paraburkholderia accounted for approximately 60%. In contrast, bulk soil isolates contained a high number of Gram-positive Firmicutes, particularly from Bacillus. Paraburkholderia accounted for ≤20% of the bacterial isolates from bulk soil, which was significantly lower than its percentage in ECM root tips. Co-cultures of six ECM fungal species with the 21 bacterial strains revealed that eight strains of three Gram-positive genera—Arthrobacter, Bacillus, and Lysinibacillus—were sensitive to the antibacterial activity of the fungi. In contrast, the Gram-negative strains, including five Paraburkholderia strains, two Burkholderia strains, and a Rhizobium sp., were not sensitive. The strength of fungal antibacterial activity varied in a species-dependent manner, but consistently affected Gram-positive bacteria. These results suggest that Gram-positive bacteria are excluded from the mycorrhizosphere by the antibacterial activity of ECM fungi, which develops specific soil bacterial communities in the mycorrhizosphere.