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Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. <i>Staphylococcus epidermidis</i>, thanks to its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in biofilm-associated infections of medical devices. Therefore, the research of new molecules able to interfere with <i>S. epidermidis</i> biofilm formation has a remarkable interest. In the present work, the attention was focused on <i>Pseudomonas</i> sp. TAE6080, an Antarctic marine bacterium able to produce and secrete an effective antibiofilm compound. The molecule responsible for this activity was purified by an activity-guided approach and identified by LC-MS/MS. Results indicated the active protein was a periplasmic protein similar to the <i>Pseudomonas aeruginosa</i> PAO1 azurin, named cold-azurin. The cold-azurin was recombinantly produced in <i>E. coli</i> and purified. The recombinant protein was able to impair <i>S. epidermidis</i> attachment to the polystyrene surface and effectively prevent biofilm formation.