The toxic influence of photosensitizers in the dark is poorly investigated. In our study we used the photosensitizers liposomal meso-tetrahydroxyphenyl chlorin derivative (Foslipos ® ) and hypericin as well as their 1:1 combination on two different head and neck squamous cell carcinoma (HNSCC) cell lines (UMB-SCC 745 and UMB-SCC 969).We examined uptake, efflux and localization of the photosensitizers with confocal microscopy. Fluorescence quantification was measured with a micro-plate spectrometer. Special interest was given to effects on cell proliferation (BrdU proliferation assay), RNA quality (Bioanalyzer measurements) and DNA damage (comet assays) in the dark.Foslipos ® uptake was linear over time and its efflux was not achieved even after 24h while uptake of hypericin reached a plateau after 5h and was almost eliminated after 24h. Localization of Foslipos ® was organelle-unspecific. Hypericin was found mainly at membranes and in trans-golgi network. Foslipos ® treated cells showed cell toxicity for the highest concentration (10μg/mL). In contrast, hypericin was toxic for all concentrations (10–0.6μg/mL). The photosensitizer combination was non-toxic for all concentrations (10–0.6μg/mL). No changes in RNA quality were monitored. Initial DNA damage was found only in hypericin treated UMB-SCC 745, which recovered after 3h. No significant DNA damage was found for UMB-SCC 969.Our data shows that the combinatorial application decrease photosensitizer toxicity, which can be advantageous in PDT treatments.