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Introduction: The establishment of a high-throughput quantification approach for waterborne pathogenic protozoa and helminths is crucial for rapid screening and health risk assessment., Methods: We developed a high-throughput quantitative polymerase chain reaction (HT-qPCR) assay targeting 19 waterborne protozoa and 3 waterborne helminths and validated its sensitivity, specificity, and repeatability. The assay was then applied to test various environmental media samples., Results: The HT-qPCR assay's limit of detection (LOD) was 5×10 2 copies/μL DNA, and its specificity was confirmed using Giardia and Cryptosporidium standards. Repeatability, assessed through intra- and inter-group experiments, yielded a coefficient of variation ( CV ) of 1.0%-4.6% and 1.2%-6.4% at concentrations of 1×10 5 and 1×10 4 copies/μL, respectively. The R 2 values of the 22 standard curves ranged from 0.983 to 0.998, with amplification efficiencies between 80% and 107%. In drinking water sources, sludge from municipal wastewater treatment plants (MWTPs), and livestock manure samples, 17 of 22 targets were detected, with Acanthamoeba genus (50.0%), Acanthamoeba castellanii (11.8%), and Enterocytozoon bieneusi (11.8%) showing high prevalence. Cryptosporidium spp., Enterocytozoon bieneusi , and Cyclospora cayetanensis were simultaneously found in all three sample types., Discussion: This study presents a useful tool for the rapid detection of waterborne protozoa and helminths in complex environmental microbiomes, providing scientific data for monitoring cross-media transmission and controlling microbial risk from a One Health perspective., Competing Interests: No conflicts of interest., (Copyright © 2025 by Chinese Center for Disease Control and Prevention.)