The aim of this study was to determine the photocatalytic effects of zinc oxide (ZnO) NPs in combination with UVA-1 in human head and neck squamous cell carcinoma (HNSCC) cell lines in vitro. NP characteristics and intracellular distribution were described by transmission electron microscopy (TEM). After pre-incubation with ZnO NPs in concentrations of 0.002-20 µg/ml, the HNSCC cell lines HLaC 78 and UD-SCC 7A as well as primary oral mucosa cells (pOMCs) were treated with UVA-1. Cell survival and vitality was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide-(MTT)-assay and fluorescein diacetate test. Apoptosis was assessed by annexin-V propidium iodide flow cytometry. Intranuclear distribution of the rod-shaped particles was observed in 3.5% of HNSCC and in 0.5% of pOMCs. UVA-1 irradiation of 15 min in combination with 0.2 and 2 µg/ml of ZnO NP dispersion was shown to reduce the vitality of cancer cell lines significantly in comparison to cells without NP exposure or UVA-1 treatment only. For HLaC 78, a significant reduction in viable cells was already seen at 10 min of UVA-1 treatment and a ZnO NP concentration of 2 µg/ml. Flow cytometry indicated that cell death occurred primarily through necrosis. In pOMCs, vitality was not influenced either by UVA-1 treatment or ZnO NP exposure up to 2 µg/ml or a combination of both. ZnO NPs showed cytotoxicity at 20 µg/ml without UVA-1. Due to their photocatalytic properties, ZnO NPs may induce cell death in human HNSCC cell lines in vitro. Further studies will evaluate a possible benefit in adjuvant cancer therapy.