Biofilm may explain the recurrences and recalcitrant episodes of otorrhea in chronic suppurative otitis media (CSOM). This study investigates bacterial biofilm in Greenlanders with CSOM and chronic otitis media with effusion (COME). The study is partly blinded, prospective and retrospective. Six children with CSOM, four children with COME and ten adults with CSOM were included in this study. Cultures were obtained and examined by standard methods. Otorrhea or glue was collected from the children and smears were prepared. Middle ear mucosa biopsies were obtained from the adults. Smears and biopsies were analyzed with microscopy and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Biofilm was confirmed in 83% of CSOM smears but in none of the COME smears. Mucosal biofilm was confirmed in 80% of the biopsies from adults with CSOM. This study provides direct morphological evidence of biofilm in samples from human CSOM. This may help to explain the microbiological mechanisms of the disease and alter the treatment strategy in the future.