Berthellina citrina is a pleurobranchid opisthobranch characterized by its skin acid-secretion that contains sulphate and chloride ions with traces of organic matter. To identify defensive potential and bioactivity, we tested this secretion for the first time against Artemia salina, different strains of microorganisms, human RBCs and human cancer cell lines. It showed lethality to A. salina with LC50 values 83.86 and 25.84 µg/ml after 6 and 24 hours, respectively and it caused 100% mortality after 48 and 72 hours. It exhibited antibacterial activity against seven human-pathogenic bacteria, with larger inhibition zone on Streptococcus pyogenes and Staphylococcus aureus and showed strong inhibition activity against seven fungal species, particularly towards Paecilomyces variotii, Aspergillus flavo-furcatis, Fusarium oxysporum and Penicillium oxalicum. It caused significant haemolysis for human RBCs in a range from 38.5 to 77.6 %. Moreover, it showed cytotoxicity against prostate carcinoma cells (PC-3), colorectal carcinoma (HCT 116) and lung carcinoma (A549) with IC50 values 6.582, 9.843 and 9.352 µg/ml, respectively. Using HPLC, taurine is the major components of the free amino acids of the secretion with percentage of 52.32%. Thus, the secretion is shown to be highly toxic, bioactive and is interpreted as a chemical defence system against natural predators, but also against fouling. In addition, it might be a new source for bioactive substances that could be used in biomedical research and drugs.