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Two different chitosan polymers were obtained from chitin extracted from the crab Polybius henslowii through different methods: (1) water-soluble chitosan (WSC) through N-acetylation with addition of acetic anhydride and (2) low molecular-weight water-soluble chitosan (LWSC) by the use of H2O2. WSC and LWSC polymers were characterized by FTIR and GPC analysis and investigated for antibacterial activity against a range of Gram-negative bacteria (Escherichia coli 1103, Escherichia coli 10503, Salmonella sp.) and Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and Lactobacillus planctarum). This study is a contribution to economic valorization of Polybius by-catch and sustainable development of west coast Portuguese fisheries communities. Financial support was obtained from European Fisheries Fund – Axis 4 managed by Fishery Local Action Group Oeste-Portugal.