AbstractAlthough the reuse of reclaimed water is one of the ways to solve the water crisis, secondary effluents still contain opportunistic pathogens that pose a potential threat to human health. Herein, we use slow filtration-disinfection as an advanced treatment to explore the removal of opportunistic pathogens such asLegionellaorPseudomonas aeruginosa. We show that, at the same filtration rate, removal is more effective (1) with aerobic heterotrophic biofilm rather than with denitrification or nitrification ones, (2) at the slowest filtration rate of 5 cm/h, and (3) when pathogens adhere to particles. According to the opportunistic pathogens content in the water after adding the disinfectant, the optimal dosage of sodium hypochlorite (NaClO) (8.0 mg/L, calculated as available chlorine concentration) and ultraviolet (UV) (40 mJ/cm2) was determined. To remove opportunistic pathogens, NaClO disinfection in slow filtration water is more effective at lower pH and higher temperature, whereas UV disinfection in slow leaching water is more effective at lower pH and turbidity. Combining slow filtration and disinfection was effective in removing opportunistic pathogens in the secondary effluent, and UV radiation was more effective than NaClO. Removal rates of Legionella and P. aeruginosa by UV exposure were 98.6% and 98.1%, respectively. Although Pseudomonas, Acidovorax, Sphingomonas, and Lactobacillus were also present in the other types of biofilms, they were more abundant in aerobic heterotrophic biofilm and had some inhibitory effect on opportunistic pathogens. Finally, turbidity, but not dissolved organic carbon (DOC), was correlated with opportunistic pathogens in water. In summary, the combined process of slow filtration-disinfection can effectively reduce the content of opportunistic pathogens in the secondary effluent and ensure microbiological safety.