The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marineBacillus licheniformisB3-15, to prevent the adhesion and biofilm formation ofPseudomonas aeruginosaATCC 27853 andStaphylococcus aureusATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA andpslAofP. aeruginosaandclfA ofS. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion ofP. aeruginosa(five logs-scale) andS. aureus(one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.