Organic micropollutants in drinking water can pose a public health risk. Chemical analysis alone cannot capture the full range of contaminants or assess their associated risks, promoting the growing use of bioanalytical tools as a complementary approach. This study assessed a drinking water treatment plant in the Nakdong River basin, Korea, using in vitro bioassays targeting nine endpoints. The highest estrogen receptor (ERα) activity was observed in the influent and significantly decreased throughout treatment. Bioactivities related to xenobiotic metabolism (PAH, PPARγ, and PXR) and oxidative stress response (Nrf2) initially increased during pre-oxidation but decreased in later treatment stages. An increase in p53 activity was also noted during treatment. Both season and treatment processes were found to affect the bioactivity variation for most endpoints, based on correlation analysis. The bioactivities observed were consistent with those reported for treated drinking waters in other countries. PAH, PPARγ, PXR, and Nrf2 activities in the final treated waters exceeded some effect-based trigger (EBT) values, indicating potential risks, although uncertainty remain regarding the EBT values for PPARγ and PXR. Additionally, the bioanalytical equivalent concentrations of volatile disinfection byproducts detected after pre- and post-chlorination were lower than the measured Nrf2 activities by factors of 7.5 and 5.5, respectively. This study highlights the importance of monitoring of bioactive chemicals to safeguard public health and ecosystems, underscoring the value of in vitro bioassays in water quality assessment.