Treated effluent from wastewater treatment plants (WWTPs) contains a diverse array of anthropogenic and natural contaminants. These contaminants have the potential to pose environmental and/or human health risks. While targeted chemical analysis is an indispensable tool for monitoring WWTP discharge, its limited chemical domain highlights the need for complementary methods. Direct toxicity assessment (DTA) using in vivo bioassays is widely used to evaluate the ecological effects and safety of effluent discharge. However, these tests are constrained by ethical considerations, specific species, high costs, low throughput and a focus on limited endpoints, such as mortality, growth and reproduction. As such, DTA does not provide a good representation of the range of sensitivities in an aquatic ecosystem. To address these challenges, in vitro bioassays have emerged as a promising alternative, offering higher sensitivity, broader toxicological coverage, faster testing and improved ethical and operational efficiency. This study investigated the potential of in vitro bioassays as a complementary approach to traditional DTA for monitoring WWTP effluent of differing treatment levels. Primary treated effluent was typically most toxic to bacteria and contained elevated levels of endocrine active compounds. WWTPs with tertiary treatment that have a combination of disinfection processes (chlorine, ozone and UV) were typically found to induce low genotoxicity. Effluent from all WWTPs induced moderate to strong aryl hydrocarbon receptor (AhR) activity along with potential nutrient-induced algal growth. A strong correlation was observed between the in vivo DTA sea urchin fertilisation test and the in vitro ERα GeneBLAzer bioassay. Of the wide range of endpoints evaluated, toxicity to bacteria, algal photosynthesis inhibition and growth, estrogenic activity and genotoxicity provided the most sensitive and relevant results. These assays have the potential to be incorporated into regulatory frameworks to supplement and screen for toxicological effects that require further investigation.