The defense system of freshwater crayfish as a diversified source of bioactive molecules with antimicrobial properties was studied. Antimicrobial activity of two polypeptide-enriched extracts obtained from hemocytes and hemolymph of were assessed against Gram positive (, and Gram negative (, ) bacteria and toward the yeast . The two peptide fractions showed interesting MIC values (ranging from 11 to 700 μg/mL) against all tested pathogens. Polypeptide-enriched extracts were further investigated using a high-resolution mass spectrometry and database search and 14 novel peptides were identified. Some peptides and their derivatives were chemically synthesized and tested in vitro against the bacterial and yeast pathogens. The analysis identified a synthetic derivative peptide, which showed an interesting antifungal (MIC and MFC equal to 31.2 μg/mL and 62.5 μg/mL, respectively) and antibiofilm (BIC equal to 23.2 μg/mL) activities against and a low toxicity in human cells.