Cystic fibrosis (CF) is one of the most common genetic lung diseases worldwide. The production of sticky viscous mucus leads to enhanced bacterial colonization and infection, but yeasts and filamentous fungi are also found abundantly in the mucus of patients suffering from CF. The role of fungi in the airways of CF patients is still not understood completely. Furthermore, recent investigations have shown that the spectrum of fungi isolated from the airways of CF patients depends strongly on the methods used. In this study, different mycological culture methods were compared: culture with a native inoculum, culture with homogenization of CF sputum, and culture after homogenization and serial dilutions of CF sputum. Altogether, 934 sputum samples from 113 patients were examined from July 2009 through December 2011. A total of 1,744 fungal isolates was recovered; 20 different yeasts and 14 filamentous fungal species were identified. Candida albicans, C. dubliniensis, and C. parapsilosis were the most common species of yeast. For the filamentous fungi, Aspergillus fumigatus was the most common, followed by Scedosporium apiospermum/Pseudallescheria boydii group and A. terreus. Many fungal, species such as Exophiala dermatitidis, Rasamsonia (Geosmithia) argillacea, and others, were isolated only from homogenized sputum samples. The longitudinal data also show that fungal colonization of CF patients is quite stable, even when treated with itraconazole. In conclusion, we recommend homogenizing CF sputa with a mucolyticum, to prepare serial dilutions, and to use appropriate fungal culture media with added antibiotics.