Abstract Foodborne bacteria biofilms present a major concern for the food industry. Although their numerous biological activities are well established, there is little research to date on the use of polysaccharides of mushroom origin as a possible solution for preventing biofilm formation. Therefore, the aim of this study was to examine the antiadhesion and antibiofilm effects of several types of Pleurotus flabellatus strain Mynuk polysaccharide extracts (PFSMpe), produced by air-lift submerged fermentation, against American Type Culture Collection (ATCC) and clinical strains of foodborne bacteria, as well as the cytotoxicity of these extracts. PFSMpe exhibited antiadhesion activity toward clinical isolates, and the percentage of adhesion inhibition was highest for water (WE) and exopolysaccharide (EXOPE) extracts (>50%) against Enterococcus faecalis. Selected ATCC strains were more resistant than clinical strains, indicating the antiquorum sensing mechanism of PFSMpe action. Antibiofilm activity was similar to antiadhesion activity and WE showed the strongest effect, again on E. faecalis. Differences in antiadhesion and antibiofilm effects of PFSMpe may be explained by differences in chemical composition, with crude extracts showing greater efficiency due to a synergistic effect. PFSMpe did not exhibit cyctotoxic activity against normal human cell lines. Overall, the findings of this study show that PFSMpe represents a promising novel strategy against bacterial biofilms.