The photoprocesses involved in the photo-induced Candida tropicalis biofilm reduction by two natural anthraquinones (AQs), rubiadin (1) and rubiadin-1-methyl ether (2), were examined. Production of singlet oxygen (1O2) and of superoxide radical anion (O2 •−) was studied. Although it was not possible to detect the triplet state absorption of any AQs in biofilms, observation of 1O2 phosphorescence incubated with deuterated Phosphate Buffer Solution, indicated that this species is actually formed in biofilms. 2 was accumulated in the biofilm to a greater extent than 1 and produced measurable amounts of O2 •− after 3h incubation in biofilms. The effect of reactive oxygen species scavengers on the photo-induced biofilm reduction showed that Tiron (a specific O2 •− scavenger) is most effective than sodium azide (a specific 1O2 quencher). This suggests that O2 •− formed by electron transfer quenching of the AQs excited states, is the main photosensitizing mechanism involved in the photoinduced antibiofilm activity, whereas 1O2 participation seems of lesser importance.