The secondary effluent of urban sewage treatment plants contains many opportunistic pathogens (OPs), which pose a potential threat to human health. In this study, the slow filtration technique is employed as the advanced treatment procedure, with the secondary effluent as the treatment object. The effectiveness of the operating conditions of the slow filtration process (filtration rate and Ca2+ concentration) in removing OPs (Pseudomonas aeruginosa, Legionella and Mycobacterium avium) and Escherichia coli from water, as well as the dynamic deposition process of pollutants on the surface of the slow filtration biofilm, was investigated. The results showed that under different filtration rates and different Ca2+ concentrations, biofilm slow filtration was more effective in removing OPs than slow filtration. The optimal filtration rate of biofilm slow filtration was 5 cm/h, and the optimal inlet Ca2+ concentration was 60 mg/L. When the filtration rate was 5 cm/h, the deposition of pollutants in the secondary effluent on the surface of biofilm slow filtration was mainly dominated by the physical adhesion of extracellular polymeric substances (EPS). When the concentration of Ca2+ was 60 mg/L, the adsorption of microorganisms was the primary method.