In this study, the putative role of phenazines and rhamnolipid-biosurfactants, antagonistic metabolites produced by Pseudomonas aeruginosa PNA1, was tested in the biological control of Pythium splendens on bean (Phaseolus vulgaris L) and Pythium myriotylum on cocoyam (Xanthosoma sagittifolium L Schott). A rhamnolipid-deficient and a phenazine-deficient mutant of PNA1 were used either separately or jointly in plant experiments. When the mutants were applied separately, no disease-suppressive effect was observed, although both mutants still produced one of the antagonistic compounds (phenazines or rhamnolipids). When the mutants were concurrently introduced in the soil, the biocontrol activity was restored to wild-type levels. Bean seeds developed significantly less pre-emergence damping-off caused by P. splendens when treated with a mixture of purified phenazine-1-carboxamide and rhamnolipids than with any of the chemicals alone. When phenazines and rhamnolipids were combined at concentrations that had no observable effects when the metabolites were applied separately, mycelial growth of P. myriotylum was significantly reduced. In addition, microscopic analysis revealed substantial vacuolization and disintegration of Pythium hyphae after incubation in liquid medium amended with both metabolites. Results of this study indicate that phenazines and biosurfactants are acting synergistically in the control of Pythium spp.