This study conducted an ecotoxicity assessment on influent samples collected from the A wastewater treatment in the Busan area, collected over a period of consecutive days. Through the identification and exploration of toxic substances in samples exhibiting toxicity, the study confirmed the presence of these toxic substances in the influent. And it investigated the impact of these toxic substances on the efficiency of the activated sludge process through Lab-scale A²O experiments. The ecotoxicity assessment using water fleas on the eight collected samples(A~H) revealed that sample A exhibited the highest toxicity. Subsequent investigation into the causative agents of toxicity identified heavy metals and ammonia as the primary contributors. Furthermore, the high ammonia concentration in influent sample A significantly affected the efficiency of activated sludge nitrification and denitrification processes. Through A²O experiments, we identified the impact of influent toxicity on the efficiency of processes utilizing activated sludge. Particularly, highly toxic influents were found to reduce the overall treatment efficiency, highlighting the necessity for influent toxicity monitoring. Therefore, this study emphasizes the importance of promptly evaluating influent toxicity and identifying toxic substances in wastewater treatment processes, highlighting the need to continuously monitor and manage key toxic substances in influent for efficient system operation.