The indolamine melatonin (MEL) is described as an antioxidant and a free radical scavenger. However occasionally, the indoleamine has been reported to increase free radicals with insufficient mechanistic explanation. In an attempt to find a reason for those controversial results, a potential mechanism that explains MEL prooxidant activity is investigated. The current controversy about redox detection methods has prompted us to search a possible interaction between MEL and dichlorodihydrofluorescein (DCFH2), perhaps the most widely fluorescence probe employed for free radicals detection in cellular models. Here, it is demonstrated that melatonin potentiates the photooxidation of DCFH2 in a cell-free system, increasing the production of its fluorescent metabolite. Indeed, MEL works as an antioxidant scavenging hydroxyl radicals in this system. Thus, this reaction between MEL and DCFH2 produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a biogenic amine with antioxidant properties too. This reaction is O2 and light dependent and it is prevented by antioxidants such as N-acetylcysteine or ascorbic acid. Furthermore, when DCFH2 has been employed to evaluate antioxidant or prooxidant activities of MEL in cellular models it is confirmed that it works as an antioxidant but these results can be modulated by light misleading to a prooxidant conclusion. In conclusion, here is demonstrated that DCFH2, light and melatonin interact and results obtained using these fluorescence probes in studies with melatonin have to be carefully interpreted.