Virulence factors (VFs) confer upon pathogens the ability to cause various types of damage or diseases. Wastewater treatment plants (WWTPs) are important point sources for the emission of pathogens and VFs into receiving rivers. Conventional WWTP upgrades are often implemented to improve the water quality of receiving ecosystems. However, knowledge on the pathogens, VFs, and health risks to receiving aquatic ecosystems after upgrade remains limited. In this study, we investigated detailed pathogenic information, including taxa, pathogenicity, and health risk, in two wastewater-dominant rivers after WWTP upgrade. Using 16S rRNA gene sequencing, we screened 14 potential pathogens in water and epilithic biofilm samples, though they were significantly more enriched in the biofilms. Combining 16S rRNA and metagenomic sequencing data, we identified Pseudomonas and Aeromonas as the dominant pathogenic taxa carrying functional VFs (e.g., mobility and offensive) in the epilithic biofilm. Moreover, strong pathogen-specific VF-host co-occurrence events were observed in the epilithic biofilm samples, indicating the importance of biofilms as reservoirs and vehicles for VFs. Further, we demonstrated that mobility VF is crucial for biofilm formation and pathogens in biofilm carrying offensive VF may be highly invasive. Quantification and health risk assessment suggested that the skin contact risk of P. aeruginosa carrying VFs was higher than the acceptable probability of 10−4 in both water and epilithic biofilm samples, which may threaten ecological and human health.