Aspergillosis is the most common and most lethal disease in captive falcons, which is most effectively treated with the poorly soluble drug Itraconazole. To obtain a high drug concentration at the side of infection, the falcon's respiratory system, an isotonic, sterile, non-toxic NLC formulation loaded with Itraconazole was developed for pulmonary application. Itraconazole-loaded NLC had a particle size well in the nanometer range and possessed a narrow particle size distribution. An entrapment efficiency of 99.98% was achieved. A good storage stability of the formulation over 6 months was found. Neither by MTS nor by LDH assay cytotoxic effects were determined on the cell line A549, indicating a good tolerability after inhalation. No physical instabilities of Itraconazole-loaded NLC could be detected nebulizing the formulation with a new nanonebulizer into a therapeutic chamber for the treatment of falcons. It could be shown, that the particle size of the aerosol generated nebulizing Itraconazole-loaded NLC with the nanonebulizer was in the nanometer range, providing the possibility to penetrate into the respiratory tract of falcons. By scintigraphy deposition of Itraconazole-loaded NLC in the lung and the air sacs of a patient was shown, being a prerequisite for the pulmonary treatment of aspergillosis in falcons.