The number of nosocomial infections related to implants and medical devices increase alarmingly worldwide. New strategies based on the design of antimicrobial coatings are required to prevent such infections. Polyelectrolyte “multilayer” films constitute a powerful tool for nanoscale surface functionalization which allows addressing this issue. By investigating films built up with poly(arginine) (PAR) of various chain lengths (10, 30, 100, and 200 residues) and hyaluronic acid (HA), we demonstrate that exclusively films constructed with poly(arginine) composed of 30 residues (PAR30) acquire a strong antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria associated with infections of medical devices. This chain-size effect is extremely striking and is the first example reported where the length of the polyelectrolytes played a key-role in the functionality of the films. Moreover, this unexpected functionality of nanolayered polypeptide/polysaccharide PAR30/HA films occurs without adding any specific antimicrobial agent, such as antibiotics or antimicrobial peptides. PAR30/HA film inhibits bacteria through a contact-killing mechanism due to the presence of mobile PAR30 chains. These chains are assumed to diffuse toward the interface, where they interact with the bacteria with the consequence of killing them. This new coating with unique properties based on the association of a homopolypeptide of 30 residues with a polysaccharide constitutes a simple system to prevent implant-related infections with a reasonable production cost.