Waterborne pathogens in urban rivers of estuarine areas pose a great threat to human health. In this study, both bacterial and fungal community structures of four urban rivers in the Pearl River estuarine area were investigated using high-throughput sequencing. Results showed that the diversity and richness indices of the bacterial community were higher than that of fungi. Taxonomic analysis indicated that the most abundant bacterial phylum was Actinobacteria, while the most abundant fungal phylum was Ascomycota. In addition, 35 major pathogens were found at the level of genus with Mycobacterium being the most abundant. Specifically, pathogens of Burkholderiales, Bartonella, Rhizobiales, Bordetella and Brucella were first detected in this area, although with relative low abundances. Spearman correlation analysis revealed that temperature, TOC and TDP were significant factors shaping both total microbial community and pathogenic microbial community structure. TDP was positively correlated with almost all the potential pathogenic genera. PICRUSt analysis further predicted that bacterial infectious disease was the most abundant pathway associated with microbial functions of human disease in all the studied rivers. This work provides useful information not only for in-depth understanding of bacterial and fungal communities in urban rivers of estuarine areas under anthropogenic disturbances but also for environmental risk management of potential waterborne pathogens.