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The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) against Candida albicans biofilms formed on two different substrates - acrylic resin or bottom of polystyrene plate; and two aPDT application regimens - twice-daily over the course of 48 h or single treatment after 48 h biofilm formation. C. albicans SN425 biofilms cultivated on Roswell Park Memorial Institute medium were incubated for 5 min with toluidine blue O (44 μM) used as a photosensitizer before red light (635 nm; 175.2 J/cm 2 ) exposure for 2 min. As negative control, ultrapure water, and as positive control 0.12% chlorhexidine (CHX) were used. Biofilms were analyzed for colony forming units (CFU) and cells morphology by confocal scanning laser microscopy. Single treatment and twice-daily aPDT on polystyrene plate and single treatment on acrylic resin did not significantly reduce the CFU (p > 0.05); in contrast, twice-daily aPDT on acrylic resin has reduced C. albicans below the detection limit, similarly to CHX treatment. Single aPDT treatment on polystyrene plate and on the resin presented a bulky and homogeneous biofilm predominantly formed by pseudohyphae. In contrast, in the resin group, the biofilm treated twice-daily with aPDT was predominantly formed by yeast cells, whilst pseudohyphae were occasionally visible. In conclusion, biofilms formed on polystyrene plates are more resistant to aPDT than biofilms formed on acrylic resin. Moreover, applying aPDT twice-daily reduces C. albicans biofilm development on acrylic resin and is a better approach against C. albicans biofilms than one single application on the mature biofilm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)