Nebulized drugs are frequently administrated through tracheostomy in clinical routine. So far, the amount of drug deposited into the lung in these patients remains unknown. The aim of our pharmacokinetic study was to compare lung delivery of amikacin in the same subjects in two settings: spontaneously breathing through a tracheostomy and through the mouth. Lung delivery was measured by amikacin urinary drug concentration in nine patients who were transitory tracheostomized for the need of a head and neck oncologic surgery. Patients performed two nebulization sessions: with a mouthpiece (MB) and through tracheostomy (TB) using a adapted jet nebulizer (Sidestream®). Lung delivery was similar with the two conditions of nebulization (6.5 ± 2.5% vs. 6.3 ± 2.0% of the nominal mass of amikacin, respectively, for MB and TB; p = 0.95). Duration of nebulization was also comparable (19.7 ± 1.6 vs. 20.1 ± 1.8 min, respectively, for mouth and tracheostomy breathing; p = 0.307). The half-life and elimination rate constant were not different between the two settings. We conclude that nebulized therapy can be administered in spontaneously breathing tracheostomized adults patients, with a similar amount of drug delivered to the lung compared with spontaneously mouth breathing patients.