A nanocomposite hydrogel based on natural polysaccharides and gold nanoparticles (ACnAu) has been prepared and its biological effects were tested in vitro with both bacteria and eukaryotic cells. Antimicrobial tests showed that AC-nAu gels are effective in killing both gram+ (Staphylococcus aureus) and gram- (Pseudomonas aeruginosa) bacteria. LDH assays pointed at a toxic effect towards eukaryotic cell-lines (HepG2 and MG63), in contrast with the case of silver-based hydrogels; cytofluorimetry studies demonstrated an apoptosis-related mechanism induced by increase of ROS intracellular level which leads to cell death after 24 h of direct contact with AC-nAu gels. In vivo biocompatibility has been evaluated in a rat model, investigating the peri-implant soft tissue reaction after 1 month of implantation. The results show that silver-containing samples induced a fibrotic capsule of the same average thickness of the control sample (devoid of nanoparticles) (∼50 μm), while in the case of gold containing materials the fibrotic capsule was thicker (∼100 μm), confirming a higher biocompatibility for silver-based samples than for gold-based ones.