Wastewater from factories producing polysulfone-based membranes mainly contains the used organic solvent, i.e., dimethylacetamide (DMAc). Due to the environmental impact of DMAc, wastewater treatment is mandatory. Several biological treatment options based on the activated sludge process are described in the literature. Due to artificial aeration, these techniques have high energy requirements. Near-nature processes such as vertical flow constructed wetlands (VF wetlands) have a low energy demand, high tolerance to load fluctuations, and low maintenance requirements. Therefore, high-loaded, two-stage VF wetlands are an efficient option for treating wastewater. However, constructed wetlands have so far only been used to a limited extent for the treatment of industrial wastewater. In the present study, the ability of laboratory-scale, high-load, two-stage VF wetlands to treat DMAc was investigated. This included their DMAc degradation efficiency and corresponding pathways, removal of the total organic carbon (TOC), nitrification and denitrification of the nitrogen, as well as the ecotoxicological effects (mutagenicity, genotoxicity, reactive oxygen species) of untreated and treated wastewater. The focus was to determine the effect of different grain size distributions on removal rates, the maximum inflow loading, and the effect of high inflow concentrations on effluent concentrations. In general, DMAc was completely degraded using VF wetlands, with dimethylamine (DMA) identified as the main intermediate. TOC removal rates reached more than 99%. The nitrogen bound to DMAc was completely nitrified. However, the start-up of the VF wetlands without seeded filter material temporarily leads to high nitrite accumulation. This may affect the mutagenicity of the treated wastewater. The results show that high-loaded, two-stage VF wetlands are an effective option for treating wastewater containing DMAc with higher efficiency than comparable biological processes.