In light of increasing resistance toward conventional antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a valuable alternative, especially for use in dentistry. In this regard, photosensitizers (PS) based on a phenalen-1-one structure seem to be especially favorable due to their high singlet oxygen quantum yield. However, the actual target structures of phenalen-1-one-mediated aPDT are still unclear. The aim of the present study was to investigate the antimicrobial efficacy of aPDT mediated by phenalen-1-one derivatives SAPYR and SAGUA for inactivation of a polymicrobial biofilm consisting of three putative periodontal pathogens in vitro and to get first insights in the mechanism of action of phenalen-1-one-mediated aPDT by assessing damage of cytoplasmic membranes. aPDT with SAPYR exhibited identical antimicrobial efficacy as compared to chlorhexidine (CHX) [4.4-6.1 log(10) reduction of colony forming units (CFUs) depending on bacterial species] while aPDT with SAGUA was less effective (2.0-2.8 log(10)). Flow cytometric analysis combined with propidium iodide (PI) staining revealed no damage of cytoplasmic membranes after aPDT with both phenalen-1-one derivatives, which was confirmed by spectroscopic measurements for release of nucleic acids after treatment. Spectrophotometric PS-uptake measurements showed no uptake of SAPYR by bacterial cells. Despite the inability to pinpoint the actual target of phenalen-1-one-mediated aPDT, this study shows the high antimicrobial potential of phenalen-1-on mediated aPDT (especially when using SAPYR) and represents a first step for getting insights in the mechanism and damage patterns of aPDT with this class of PS.