Melatonin (MLT), N-acetyl-5-methoxytryptamine, is mainly secreted by the pineal gland. The ultraviolet (UV), infrared (IR) and 1H-NMR spectra of irradiated and non-irradiated MLT were measured, and phototoxicity tests of MLT, anthracene (positive control) and sodium lauryl sulfate (SLS, negative control) were performed. The methods employed include both in vitro tests such as MTS assay using the human fibroblast cell and yeast growth inhibition assay using Candida albicans and in vivo method using the skin of guinea pig. UV absorption spectra and 1H-NMR spectra of MLT were changed by UVA (365 nm, 15 J/cm2), but IR spectra of MLT were not changed. The fifty percent inhibitor concentration (IC50) ratio (UV-/UV+) of MLT was 10. The inhibition zone of irradiated-paper disks treated with MLT was not observed. According to the results of histopathological examination, no pathologic lesion was observed in the non-irradiated group, but slight degeneration of keratinocytes in the epidermis, hemorrhage and vasodilation in dermis were observed in the irradiated group. These results indicate that the molecular structure of MLT is altered by UVA to unidentified photoproducts and a moderate phototoxicity of MLT is predicted.