The identification of the priority control sequence of pollutants in effluents of wastewater treatment plants (WWTPs) has important implications for the management of water quality. This study chose 34 typical pollutants based on their representativeness and detection rates in municipal wastewater. The occurrence frequency and concentration of these pollutants in 168 Chinese WWTP effluents were measured at the national level. The data on in vitro toxicity (67 assays) and in vivo toxicity (216 species) for target pollutants were obtained from the public toxicity database and our experimental data. An environmental health prioritization index (EHPi) method was proposed to integrate the occurrence frequency, concentration, removal rate, and in vitro and in vivo toxicity to determine the priority control sequence of target pollutants. Ethynyl estradiol, 17β-estradiol, estrone, diclofenac, and atrazine were the top 5 pollutants identified by the EHPi score. Several pollutants with high EHPi scores showed spatial differences. Besides the EHPi method which was from the single pollutant perspective, the combined toxicity of pollutants (300 pairs of binary combinations) was also measured based on in vitro toxicity assays to evaluate the key pollutants from the pollutant-pollutant interacting perspective. The pollutants (such as ofloxacin and acetaminophen) that could have significant synergetic effects with many other pollutants are worthy of prior attention. This study shed new light on the identification of the priority control sequence of pollutants in WWTP effluents. The results provide meaningful data for the effective management and control of wastewater water quality.