The antifungal compound 2,4-diacetylphloroglucinol (Phl) contributes to biocontrol in pseudomonads, but whether or not Phl(+) biocontrol pseudomonads display higher plant-protecting activity than Phl(-) biocontrol pseudomonads remains to be demonstrated. This issue was addressed by assessing 230 biocontrol fluorescent pseudomonads selected from a collection of 3132 bacterial isolates obtained from 63 soils worldwide. One-third of the biocontrol pseudomonads were Phl(+) and almost all Phl(+) isolates also produced hydrogen cyanide (HCN). The only Phl(+) HCN(-) strain did harbor hcn genes, but with the deletion of a 134 bp hcnC fragment corresponding to an ADP-binding motif. Statistical analysis of biocontrol isolate distributions indicated that Phl production ability was associated with superior disease suppression activity in the Pythium-cucumber and Fusarium-tomato pathosystems, but this was also the case with HCN production ability. However, HCN significance was not as strong, as indicated both by the comparison of Phl(-) HCN(+) and Phl(-) HCN(-) strains and by correlation analyses. This is the first population-level demonstration of the higher plant-protecting activity of Phl(+) biocontrol pseudomonads in comparison with Phl(-) biocontrol pseudomonads.