Urban villages are unique residential neighborhoods in urban areas in China. Roof tanks are their main form of water supply, and water quality deterioration might occur in this system because of poor hygienic conditions and maintenance. In this study, water samples were seasonally collected from an urban village to investigate the influence of roof tanks as an additional water storage device on the variation in the microbial community structure and pathogenic gene markers. Water stagnation in the roof tank induced significant decreases in chlorine (p < 0.05), residual chlorine was as low as 0.02 mg/L in spring. Propidium monoazide (PMA)-qPCR revealed a one-magnitude higher level of total viable bacterial concentration in roof tank water samples (2.14 ± 1.81 × 105 gene copies/mL) than that in input water samples (3.57 ± 2.90 × 104 gene copies/mL, p < 0.05), especially in spring and summer. In addition, pathogenic fungi, Mycobacterium spp., and Legionella spp. were frequently detected in the roof tanks. Terminal users might be exposed to higher microbial risk induced by high abundance of Legionella gene marker. Spearman's rank correlation and redundancy analysis showed that residual chlorine was the driving force that promoted bacterial colonization and shaped the microbial community. It is worth noted that the sediment in the pipe will be agitated when the water supply is restored after the water outages, which can trigger an increase in turbidity and bacterial biomass. Overall, the findings provide practical suggestions for controlling microbiological health risks in roof tanks in urban villages. Display omitted