OBJECTIVES: Demonstrate that bacterial biofilm in sinus mucosal samples from patients with eosinophilic mucin chronic rhinosinusitis (EMCRS) and allergic fungal rhinosinusitis (AFRS) contains fungal elements; identify specific organisms in the biofilm. METHODS: Mucosa samples from 11 patients undergoing sinus surgery were collected. Patients were classified as having AFRS, EMCRS, or chronic rhinosinusitis (CRS) based on histopathologic findings. Three mucosal samples from controls were also collected. Samples were stained with specific bacterial fluorescent in situ hybridization (FISH) DNA probes (Haemophilus influenzae, Streptococcus pneumophilia, Staphylococcus aureus, and Pseudomonas aeruginosa) and a general pan-fungal FISH probe. The samples were analyzed for bacterial biofilm ultrastructure and fungal elements using epifluorescent microscopy. RESULTS: Bacterial biofilm was demonstrated in 9/11 samples and 2/3 controls. H. influenzae was the predominant biofilm present. There was a trend showing more fungal elements in AFRS and EMCRS biofilms than in CRS and controls. CONCLUSION: This is a preliminary study demonstrating fungal elements within sinus mucosal biofilm and demonstrating biofilm in AFRS.