In this work we study (i) the formation and stabilization of silver nanoparticles in a bioactive chitosan-derived polysaccharide solution, (ii) the antimicrobial properties, either in solution or in 3D hydrogel structures, obtained by mixtures with the polysaccharide alginate, and (iii) the cytotoxicity of the latter nanocomposite materials on different eukaryotic cell lines. Antimicrobial results show that these nanocomposite systems display a very effective bactericidal activity toward both Gram+ and Gram- bacteria. However, the hydrogel does not show any cytotoxic effect toward three different eukaryotic cell lines. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. This novel finding could advantageously contribute to responding to the growing concerns on the toxicity of nanoparticles and facilitate the use of silver-biopolymer composites in the preparation of biomaterials.