Intensive agriculture activities, industrialization and growing numbers of wastewater treatment plants along river banks collectively contribute to the elevated levels of neurotoxic pollutants in natural water reservoirs across Europe. We established an in vitro bioassay based upon neural stem cells isolated from the subventricular zone of the postnatal mouse to evaluate the neurotoxic potential of raw wastewater, treated sewage effluent, groundwater and drinking water. The toxic potential of water samples was evaluated employing viability, proliferation, differentiation and migration assays. We found that raw wastewater could reduce the viability and proliferation of neural stem cells, and decreased the neuronal and astrocyte differentiation, neuronal neurite growth, astrocyte growth and cell migration. Treated sewage water also showed inhibitory effects on cell proliferation and migration. Our results indicated that relatively high concentrations of nitrogenous substances, pesticides, mercuric compounds, bisphenol-A, and phthalates, along with some other pollutants in raw wastewater and treated sewage water, might be the reason for the neuroinhibitory effects of these water samples. Our model successfully predicted the neurotoxicity of water samples collected from different sources and also revealed that the incomplete removal of contaminants from wastewater can be problematic for the developing nervous system. The presented data also provides strong evidence that more effective treatments should be used to minimize the contamination of water before release into major water bodies which may be considered as water reservoirs for human usage in the future.