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Fusarium Head Blight (FHB) caused by Fusarium graminearum pathogens constitutes a major threat to agricultural production because it frequently reduces the yield and quality of the crop. The disease severity is predicted to increase in various regions owing to climate change. Integrated management where biocontrol plays an important role has been suggested in order to fight FHB. P. polymyxa A26 is known to be an effective antagonist against F. graminearum. Deeper understanding of the mode of action of P. polymyxa A26 is needed to develop strategies for its application under natural settings in order to effectively overcome the pathogenic effects. This study aims to re-evaluate a former study and reveal whether compounds other than non-ribosomal antibiotic lipopeptides could be responsible for the antagonistic effect, despite what is often reported. Wheat seedlings were grown to maturity and the spikes infected with the pathogen under greenhouse conditions. The development of FHB infection, quantified via the disease incidence severity and 100-kernel weight, was strongly correlated (r > 0.78, p < 0.01) with the content of the polysaccharide component D-glucuronic acid in the biofilm. Furthermore, while increased inoculum density from 10(6) to 10(8) cells/ml did not affect wild type performance, a significant increase was observed with the P. polymyxa mutant deficient in nonribosomal lipopeptide synthesis. Our results show that P. polymyxa A26 biofilm extracellular polysaccharides are capable of antagonizing F. graminearum and that the uronate content of the polysaccharides is of critical importance in the antagonism.