BACKGROUND: A polymerase chain reaction (PCR)-based method has been used to identify oral anaerobic pathogens in biofilms on voice prostheses. The purpose of the present study was to determine the location of those pathogens inside the biofilms. METHODS: Biofilms of 15 voice prostheses were sampled and used to identify the oral pathogens. Fluorescence in situ hybridization was applied on smears made on glass slides and on sections of intact biofilms visualized by confocal laser scanning microscopy (CLSM). RESULTS: Fusobacterium nucleatum (F. nucleatum) was the most frequently detected pathogen and the only tested species detected in microcolonies. The other microbes (Parvimonas micra [P. micra], Porphyromonas gingivalis [P. gingivalis], Tannerella forsythia [T. forsythia], and Treponema denticola [T. denticola]) were not detected or only detected as single cells. CLSM analysis showed that F. nucleatum resided on the biofilm surface. CONCLUSION: Although detectable, oral anaerobic pathogens seem to be no more than passers-by that adhere without further observed proliferation and apparently play no striking role in biofilm formation on voice prostheses.