To evaluate the antimicrobial, hemolytic and cytotoxicity of toxin present in the puffer fish Arothron hispidus from the southeast coast of India. Liver, Skin, Muscle tissue extract of A. hispidus was extracted by using the acetic acid. In vitro antimicrobial test was done by disc diffusion method against seven bacterial and five fungal strains. Hemolytic and cytotoxicity test against HeLa cell line done by following the standard method. All the extract has shown inhibitory activity for antimicrobial test against bacterial and fungal strains, the skin extract has shown maximum zone against the E. coli and A.niger and the liver extract has shown minimum against P. vulgaris and T. viridae. Maximum hemolytic activity was observed in the skin extract of A. hispidus. In cytotoxic test, the maximum level of inhibition was observed on the growth of Hela 2 cell line by the skin extract of fish. The IC 50 value of skin extract was found to be 1.78 mg/ml. These results indicated that the skin extracts of A. hispidus may have highly biologically active compound that can be further explored and utilized for welfare of mankind. © 2013 IJDDR, Samanta S Khora et al.