Abstract: Polysaccharide isolated from the cuttlebone of Sepia aculeata and Sepia brevimana using 10 mM EDTA were studied for their antibacterial and antifungal activity against nine bacterial (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhii and Shigella sp.) and four fungal (Candida sp., Rhizopus sp., Aspergillus flavus and Aspergillus fumigatus) pathogens at different concentrations such as 25, 50, 75 and 100% against control. The activities were found to be increasing with the increasing concentration of the extracts. No antibacterial activity was recorded against V. cholerae in all concentrations of S. brevimana. In S. aculeata, maximum (8 mm inhibition zone in 100%) and minimum (2 mm inhibition zone in 25%) activity was recorded against E. coli, but in S. brevimana the highest and lowest activity was recorded as 17 mm (100%) and 2 mm (25%) (inhibition zone) against P. aeruginosa and E. coli, respectively. In the antifungal activity study, the highest and lowest inhibition zones of 12 mm (100%) and 3 mm (25%) were noted against A. flavus, Candida sp. and A. fumigatus and Rhizopus sp. respectively, but in S. brevimana, maximum and minimum activity of 9 mm (100%) and 2 mm (25%) (inhibition zone) were observed against A. flavus and A fumigatus, respectively. In both species the cuttlebone polysaccharide showed no activity against Candida sp.