Oral and middle ear infections are biofilm related infections. Fusobacterium nucleatum is considered to be a key species in dental biofilm formation. The aim of this study was to compare conventional and real-time PCR, for the detection of F. nucleatum in saliva, nasopharyngeal secretion and middle ear effusion samples. Sixty samples were collected from 20 patients with otitis media with effusion. Species-specific 165 rRNA primers were used for conventional PCR and species-specific primers and TaqMan probes were used for real-time PCR analysis. Real-time PCR had a better performance than conventional PCR in detecting bacteria in the analyzed samples. The tested real-time PCR assay can identify) and identify) F nucleatum in biofilm samples with a high degree of sensitivity when compared with conventional PCR techniques.