A constant increase of nosocomial infections that are caused by adhesion and colonization of pathogenic microorganisms, especially drug-resistant bacteria, on the surfaces of healthcare devices has received considerable attention worldwide. In this study, bioinspired by antimicrobial skins of natural living beings, we developed a self-healing bactericidal coating through the immobilization of hydrophobic antimicrobial peptides (AMPs) into a multilayer film, which was constructed through the enhanced exponential layer-by-layer assembly of polyethylenimine (PEI) and poly(acrylic acid) (PAA). The (PEI/PAA) film shows particular dynamic properties from the as-prepared thin solid film to a spongy microporous structure via acid solution treatment, and then back to the thin solid film by eliminating micropores via the treatment of saturated humidity. Consequently, the loading and integration of hydrophobic AMPs such as gramicidin A (GA) into the (PEI/PAA) film were achieved via simple wicking action with GA solution and subsequent humidity treatment, respectively. The GA loading densities can be precisely controlled by using different concentrations of GA solution. We demonstrated that the GA immobilized (PEI/PAA) film has rapid self-healing properties, and that Gram-positive bacteria S. aureus including the methicillin-resistant type were efficiently killed through the contact-killing mode. Collectively, this self-healing bactericidal coating shows practical potential in a variety of healthcare applications.