The proliferation and chlorine resistance of pathogenic bacteria in drinking water distribution systems (DWDSs) pose a serious threat to human health. In this study, the synergistic effects of ozonation pretreatment and trace phosphate on water quality health risk and microbial stability were investigated in the small-scale DWDSs simulated by biofilms annular reactors with cast iron coupons. The results indicated that ozonation of drinking water containing trace phosphate was equivalent to increasing microbial carbon and phosphorus sources, further leading to the rapid proliferation of opportunistic pathogens (OPs) in subsequent DWDSs. Under the influent condition of ozonation pretreatment and 0.6 mg/L phosphate, the gene copy numbers of living Legionella spp., Mycobacterium spp., and Acanthamoeba spp. reached up to 1.50 × 10