Aspergillus fumigatus is often found in the respiratory tract secretions of patients with cystic fibrosis (CF), although the role of the fungus for progression of pulmonary disease remains unclear. This study aimed to investigate the frequency of A. fumigatus and other fungi in sputum of adult CF patients using different methods for culture and microscopy. Results from the analysis of 369 samples from 94 patients showed that A. fumigatus could be isolated in 45.7% of patients. Other moulds were rare, but the yeast Candida albicans was another frequent isolate, detected in 75.5% of patients. A comparison of different culture media showed no difference between a selective medium developed to specifically inhibit Pseudomonas aeruginosa and a standard fungal culture medium for growth of A. fumigatus, although both were more efficient for detection of fungi than other bacterial culture media. Fluorescent microscopy with calcofluor white was more sensitive for detection of fungal hyphae in undiluted sputum than standard methylene blue staining. This study shows that A. fumigatus and C. albicans have a high frequency in adult CF patients. Microbiological analysis should routinely include methods for specific identification of fungi to monitor for potential complications arising from fungal disease in these patients.