Wastewater discharge, urban run-off, and agricultural activities are adversely affecting the quality of waterways globally. These activities release complex mixtures of elemental and organic contaminants including pharmaceuticals, industrial compounds, and agrichemicals, which pose environmental risks. Monitoring water and sediment quality using targeted chemical analysis of known chemicals of concern cannot account for the effects of all chemicals present in mixtures. Effect-based methods (EBM) using in vitro bioassays can provide high-level assessments of organic micropollutants in samples by measuring the total activity associated with all bioactive chemicals, overcoming some drawbacks associated with targeted chemical analysis. This study applied a battery of cell-based bioassays for 12 endpoints, including bacteria toxicity, algal and mammalian cells, genotoxicity, AhR activity, photosynthesis inhibition, and endocrine activity in water passive sampler and sediment extracts collected from urban and agricultural catchments in New Zealand. Bioassay results were expressed as toxic units, genotoxic units, and bioanalytical equivalent concentration (BEQ), and compared with available effect-based trigger values (EBTs) to estimate overall quality. Bioassay responses exceeded EBTs at multiple locations, with some exceeding EBT values by a factor of 10 or more. Notably, extracts from some sites produced significant estrogenic activity, which is commonly found in wastewater discharge and agricultural runoff. Herbicidal activity was observed in Southland samples at concentrations indicative of a risk to photosynthetic organisms. Hotspots of toxicity to bacteria and genotoxicity were noted across sampling sites, and AhR activity was seen across both catchments, predominately at downstream sites where urbanisation begins to predominate catchment land use. Sample sites adjacent to landfill sites exhibited significant activity across most bioassays, highlighting the importance of proper landfill management and the risk that historical closed landfills continue to present in the environment. Going forward, we plan to combine effect-directed analysis with non-target analysis to identify chemicals of emerging concern in New Zealand.