Prevention of pathogen colonization of medical implants is a major medical and financial issue since infection by microorganisms constitutes one of the most serious complications after surgery or critical care. Immobilization of antimicrobial molecules on biomaterials surfaces is an efficient approach to prevent biofilm formation. Herein, the first self-defensive coating against both bacteria and yeasts is reported, where the release of the antimicrobial peptide is triggered by enzymatic degradation of the film due to the pathogens themselves. Biocompatible and biodegradable polysaccharide multilayer films based on functionalized hyaluronic acid by cateslytin (CTL), an endogenous host-defensive antimicrobial peptide, and chitosan (HA-CTL-C/CHI) are deposited on a planar surface with the aim of designing both antibacterial and antifungal coating. After 24 h of incubation, HA-CTL-C/CHI films fully inhibit the development of Gram-positive Staphylococcus aureus bacteria and Candida albicans yeasts, which are common and virulent pathogens agents encountered in care-associated diseases. Hyaluronidase, secreted by the pathogens, leads to the film degradation and the antimicrobial action of the peptide. Furthermore, the limited fibroblasts adhesion, without cytotoxicity, on HA-CTL-C/CHI films highlights a medically relevant application to prevent infections on catheters or tracheal tubes where fibrous tissue encapsulation is undesirable.