Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis. 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 μg/μl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Seppak C-18® 20% acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules.