Municipal and Industrial Solid Waste Incineration (MISWI) bottom ash is mainly deposited in landfills, but natural resources and energy could be saved if these ash materials would be used in geotechnical constructions. To enable such usage, knowledge is needed on their potential environmental impact. The aim of this study was to evaluate the ecotoxicity of leachates from MISWI bottom ash, aged for five years, in an environmental relevant way using a sequential batch leaching method at the Liquid/Solid-ratio interval 1–3, and to test the leachates in a (sub)chronic ecotoxicity test. Also, the leachates were characterized chemically and with the technique of diffusive gradients in thin films (DGTs). By comparing established ecotoxicity data for each element with chemically analysed and labile concentrations in the leachates, potentially problematic elements were identified by calculating Hazard Quotients (HQ). Overall, our results show that the ecotoxicity was in general low and decreased with increased leaching. A strong correspondence between calculated HQs and observed toxicity over the full L/S range was observed for K. However, K will likely not be problematic from a long-term environmental perspective when using the ash, since it is a naturally occurring essential macro element which is not classified as ecotoxic in the chemical legislation. Although Cu was measured in total concentrations close to where a toxic response is expected, even at L/S 3, the DGT-analysis showed that less than 50% was present in a labile fraction, indicating that Cu is complexed by organic ligands which reduce its bioavailability.