The microbiota of the denture plaque biofilm colonizing the fitting surface of dentures in edentulous subjects with healthy palates (n = 20) and in edentulous subjects with denture stomatitis (n = 20) was studied. The numbers of bacteria colonizing the dentures of healthy subjects was significantly less than the numbers colonizing the dentures of stomatitis subjects. The proportions and frequency of isolation of mutans streptococci, lactobacilli, bifidobacteria and yeasts were significantly (P < 0.05) greater in the subjects with denture stomatitis. The proportions of these organisms in the denture plaque biofilm of the subjects with denture stomatitis were similar to those found in carious lesions, indicating that the site is a low pH environment. The predominant bifidobacterial species in the mouths of dentate subjects is Bifidobacterium dentium but in the edentulous subjects wearing dentures B. dentium was isolated from only one of the 20 subjects with denture stomatitis and from none of the 20 subjects with healthy palates. Instead, Bifidobacterium breve, Bifidobacterium scardovii and Bifidobacterium longum subsp. longum were isolated. Only a single non-oral bifidobacterial species was isolated from each individual and repetitive extragenic palindromic- and BOX-polymerase chain reaction typing methods indicated that the same genotypes were shared between subjects. Using deferred antagonism spot plate assays, interspecies inhibition was demonstrated between oral isolates of B. dentium, B. breve, B. scardovii and B. longum subsp. longum. Here we have shown that bifidobacteria and caries-associated microbiota are present in denture plaque at levels similar to those of carious lesions and B. dentium cannot be maintained in an edentulous mouth.