The adhesion of bacteria is one of the most important stages in biofilm formation and bacterial infection. Once bacteria have adhered to a biomaterial surface, it is hard to eliminate them, and bacterial growth and infection are inevitable. In the current study, we have designed and constructed enzymatically degradable (hyaluronic acid/chitosan)n–(hyaluronic acid/polylysine)n ((HA/CHI)n–(HA/PLL)n) composite multilayer films via a layer-by-layer self-assembly method. Spectroscopic ellipsometry and scanning electron microscopy cross section measurements showed the exponential growth behavior of (HA/CHI)10–(HA/PLL)10 multilayer films (∼2 μm). The increased secretion of hyaluronidase and chymotrypsin in the bacterial infection microenvironment led to the fast degradation of the outer (PLL/HA)n multilayer films in the first 24 h. Enzymatic degradation of the multilayer films efficiently reduced the adhesion of both Staphylococcus aureus and Escherichia coli (>99%). Bacterial live/dead staining demonstrated the bactericidal action of the remaining bottom (CHI/HA)n multilayer films against the two kinds of bacteria. In vivo subcutaneous tests on New Zealand white rabbits, wound appearance and histopathology analysis showed that the implantation of composite multilayer film-modified PDMS promoted wound healing and the materials demonstrated a self-defense antibacterial effect. The material demonstrated both anti-adhesive and bactericidal properties and could be used to modify biomedical implants.