A proportion of patients with chronic rhinosinusitis, especially if nasal polyps are present, have a diagnosis of fungal rhinosinusitis. The diagnosis is difficult to establish because the symptoms and clinical and radiological signs are non-specific. Also current diagnostic methods, i.e. histology, fungal staining and culture, are insensitive. The performance of the Aspergillus galactomannan (GM) ELISA and real-time PCR for Aspergillus fumigatus mitochondrial DNA was evaluated for the detection of Aspergillus in sinus mucus samples from 25 patients with chronic rhinosinusitis with nasal polyposis. The results were compared with those from nasal lavage fluid from 19 healthy volunteers. Seven patients (28 %) were diagnosed as having fungal rhinosinusitis according to the presence of filaments in histology or direct microscopy using Calcofluor white. All fungal rhinosinusitis patients were negative in the GM ELISA. GM ELISA was positive in five patients whose samples were negative using conventional methods and A. fumigatus PCR. Two out of seven patients with fungal rhinosinusitis were positive by A. fumigatus PCR: one also had a positive A. fumigatus culture, and one had hyphae consistent with Aspergillus in histology. One additional patient had a weak positive PCR result, but other fungal tests were negative. In control subjects, the GM ELISA was positive in 21 %, whereas direct microscopy, culture and A. fumigatus PCR were negative in all samples. Direct microscopy and culture together with histology remain pivotal in defining fungal rhinosinusitis diagnosis. A. fumigatus PCR may have additional value in allowing the diagnosis to be made sooner, whereas the GM ELISA is not reliable in diagnosing Aspergillus infection of the paranasal sinuses.