5,6-Dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, the aim of this study was to assess possible toxic effects of DHI on cells related to the eye, such as human ARPE-19 cells and mouse retinal explants. Moreover, DHI was tested on its effects on retinal function in vivo using electroretinography. We found cytotoxicity of DHI against ARPE-19 cells at 100 µM, but not at 10 µM. 10 µM DHI exhibited a slight, though not significant protective activity against UV-A damage in ARPE-19 cells. We found cytoprotection in cultured mouse retinas by 50 µM DHI or its diacetylated derivative 5,6-diacetoxyindole (DAI), respectively. In ERG measurements in vivo, amplitudes were decreased only slightly by 100 µM DHI compared to saline, whereas a better preservation of amplitudes was visible at 10 µM DHI, in particular with respect to cones. In histological sections, more cones were found at 10 µM DHI than at 100 µM DHI. As a conclusion, DHI shows a slight protective effect at 10 µM both in vitro and in vivo. At 100 µM, it shows a strong cytotoxicity in vitro, which is strongly reduced in vivo.