Aims: Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system. Methods and Results: We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253 center dot 7 to 15 center dot 8 mg ml-1. We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31 center dot 7 mg ml-1) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited. Conclusions: The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9-41 of a beta-thymosin of P. lividus. Significance and Impact of the Study: The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms.