Polyurethane (PU) substrates are biocompatible materials widely used to manufacture endotracheal tubes. However, in common with other biomedical materials, they are liable to the formation of microbial films. The occurrence of pneumonia in intubated patients treated at intensive care units often takes the form of ventilator-associated pneumonia (VAP). The issue relates to the translocation of pathogenic microorganisms that colonize the oropharyngeal mucosa, dental plaque, stomach, and sinuses. New protective materials can provide a more effective therapeutic approach to mitigating bacterial films. This work concerns microcrystalline carbon film containing dispersed silver nanoparticles (μC-Ag) deposited on PU substrates using a physical vapor deposition sputtering process. For the first time, carbon paper was used to produce a carbon target with holes exposing a silver disk positioned under the carbon paper, forming a single target for use in the sputtering system. The silver nanoparticles were well distributed in the carbon film. The adherence characteristics of the μC-Ag film were evaluated using a tape test technique, and electron dispersive x-ray mapping was performed to analyze the residual particles after the tape test. The microbicidal effect of the thin film was also investigated using species S. aureus, a pathogenic microorganism responsible for most infections of the lower respiratory tract involving VAP and ventilator-associated tracheobronchitis (VAT). The results demonstrated that μC-Ag films on PU substrates are promising materials for mitigating pathogenic microorganisms on endotracheal tubes.