Microbial infection is one of the most serious problems in the field of medical devices, particularly in implants. Herein, we have designed and constructed a (mucin/poly(ethyleneimine))n ((mucin/PEI)n) multilayer film using a layer-by-layer self-assembly method and the grafting of antimicrobial peptides to enhance the bactericidal efficacy. Water contact angle measurements and atomic force microscopy images revealed that the hydrated multilayer film created a highly hydrophilic surface with a low roughness. The functionalized polydimethylsiloxane (PDMS) surfaces were shown to be effective in preventing bovine serum albumin (BSA) adsorption and in reducing bacterial adhesion. Bactericidal activity of the (mucin/PEI)n-nisin coatings, measured by scanning electron microscopy and a LIVE/DEAD bacterial viability kit, was remarkably effective against S. aureus owing to the grafting of nisin. In vivo subcutaneous incisions were made in New Zealand white rabbits and were implanted with multilayer-film-modified and uncoated PDMS. Both the evaluation of the appearance of the wound and the histopathology analysis demonstrated that implantation of the antibacterial-coating-modified PDMS promoted wound healing and showed an anti-inflammatory effect. The multilayer film proved to be nontoxic towards human lens epithelial cells, which can potentially be widely used to modify biomedical implants.