Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.