Quaternary ammonium compounds (QAC) are widely used as disinfectants, biocides, and detergents, among a variety of other applications. The cationic surface-activity of QAC determines their potential to act as a biocide on both target and non-target organisms. This study aims to provide a broad-based environmental risk characterization and evaluation for selected QAC with particular focus on the situation in Austrian rivers. A modular study design was employed involving environmental exposure characterization, QAC fate and effect analysis in wastewater, ecotoxicological effect characterization, and environmental risk evaluation. A wide array of Austrian surface waters and wastewater effluents were screened for the selected key compounds, benzalkonium chlorides and dialkyldimethylammonium chlorides with different C-chain lengths. Ecotoxicological effect characterization was based on both microbiotests for a set of representative aquatic organisms and a literature review. For risk evaluation, the PEC/PNEC ratio was above 1 for some rivers. Notably, small rivers with a high particulate matter were contaminated with QAC in the case of high water incidents. Hence, for the Austrian sites studied, a QAC-derived risk to sensitive aquatic non-target organisms could not be excluded.