A number of gram-negative bacteria have a quorum-sensing system and produce the N-acylhomoserine lactone (AHL) as a signal molecule. Pseudomonas chlororaphis subsp. aurantiaca StFRB508 produces one of the phenazine derivatives, phenazine-l-carboxylic acid (PCA). Whole-genome sequencing of StFRB508 revealed the presence of two sets of AHL-synthase and AHL-receptor gene, phzIR and aurIR. The mutation of phzI drastically decreased PCA production, but the mutation of aurI did not affect PCA production. The phzI and awl double mutant did not show any PCA production. StFRB508 produces three major AHLs, N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). As the results of TLC analysis, PhzI mainly catalyzes the biosynthesis of 3-hydroxy-C6-HSL, and AurI catalyzes the biosynthesis of C4-HSL and C6-HSL. PCA production in the phzI and aurI double mutant was restored by exogenous AHLs and the most active AHL was 3-hydroxy-C6-HSL. StFRB508 showed high inhibitory activity of the development of mycelia of plant pathogenic fungi, Fusarium oxysporum f. sp. conglutinans. However, the phzI and mid double mutant could not inhibit the development of mycelia. These results demonstrated that the multiple quorum-sensing system play an important role in PCA production and antifungal activity in StFRB508. 2013, The Society for Biotechnology, Japan. All rights reserved.