Burkholderia ambifaria BC-F, a biocontrol strain reported previously to exhibit broad-spectrum antifungal activity, was highly active in formation of N-acyl homoserine lactones (AHLs). We constructed AHL-deficient derivatives of strain BC-F in which the genes specifying AHL synthase (bafI) and AHL-binding transcriptional activator (bafR) were inactivated by allelic exchange. The resulting AHL-deficient mutants had decreased antifungal activity.