Objective: To evaluate the antimicrobial activity of polysaccharide from cuttlebone and methanolic extract from body tissue of Sepia prashadi, against ten human pathogenic bacteria and five fungi. Methods:The activity of polysaccharide and methanolic extract was investigated against Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Vibrio alginolyticus, Staphylococcus aureus, Vibrio parahaemolyticus, Streptococcus sp., Streptococcus pneumoniae, Salmonella sp. and Escherichia coli, and five fungal strains such as Alternaria alternata, Candida tropicalis, Penicillium italicum, Fusarium equiseti and Candida albican using disc diffusion method and minimum inhibitory concentration (MIC) were also calculated. Results:Both polysaccharide and methanolic extract was active against gram positive than that of gram negative pathogenic bacteria but inactive against fungi. The MIC of both the extract ranging from 60 to 100 mg/mL. Conclusions: These results suggest that cephalopod polysaccharide and methanolic extract possess relatively good antibacterial activity.