Red autofluorescence of plaque and its relation to fluorescence of a single species in the biofilm was studied. Fluorescence images of non-disclosed and disclosed plaque of 28 first-year students were captured. The plaque samples were assessed by culture methods and studied for red autofluorescence. Species capable of producing red autofluorescence were cultivated from subjects with and without red plaque autofluorescence. Red autofluorescence was observed from Actinomyces odontolyticus, Prevotella intermedia and from Porphyromonas gingivalis and Peptostreptococcus micros grown together. The microbial findings indicated that the intrinsic characteristics of the mature biofilm itself are more responsible for the red autofluorescence than the characteristics of the single species.