The aim of this study was to determine the microbiological origin of plaque fluorescence observed during quantitative light-induced fluorescence (QLF) analysis. Plaque was sampled from dentures, because of easy accessibility and the homogeneous background provided by the denture tooth during imaging, and the acknowledged comparability to occlusal plaque. Forty removable poly(methyl methacrylate) dentures were screened for the presence of fluorescent plaque deposits during QLF analysis. Dentures were photographed, QLF images were recorded and samples of fluorescent plaque were taken. Plaque samples were cultured on fastidious anaerobe agar, Wilkins Chalgren agar and Sabourauds dextrose agar. Plates were screened under QLF and fluorescent colonies were subcultured and identified. Areas of red, orange and green fluorescence were detected on the fitting and non-fitting surfaces of dentures. The red and orange fluorescing species were Prevotella melaninogenica, Actinomyces israelii and Candida albicans, which are generally acknowledged to be secondary colonisers, present in more mature plaque. Green fluorescence was observed in streptococcal species (early colonisers) and Fusobacterium nucleatum (important organism in plaque development). Non-fluorescent colonies were also cultured. Plaque which accumulates on susceptible surfaces tends to be associated with caries, but it may be its maturity, rather than the presence of cariogenic streptococci, that is more likely to provide a microbiological link between red fluorescence and caries.