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Centralized water supply in rural areas, supported by small waterworks (small-central mode) and by municipal water treatment plants (urban-extension mode), is an important guarantee to implement the Rural Revitalization Strategy Plan (2018–2022) in China. Opportunistic pathogens (OPs) could not be evaluated by the national drinking water sanitation standards in China (GB 5749–2022), posing potential microbial risks in rural drinking water. In this study, the spatiotemporal distribution of OPs, microbial community and the associated functional composition under two central water supply modes were investigated by molecular approaches. The results indicated that OPs were widely presented in the rural drinking water regardless of water supply modes, and were more abundant than those in the urban tap water. The insufficient residual chlorine and higher turbidity triggered more microbial proliferation, posing a seasonal variation of OPs gene copy numbers and bacterial community compositions. In warm seasons of summer and autumn, the gene copies of E. coli , M. avium , Pseudomonas spp. and the amoeba host Acanthamoeba spp. achieved up to 4.92, 3.94, 6.75 and 3.74 log 10 (gene copies/mL), respectively. Potential functional prediction indicated higher relative abundance of pathogenic genes and infectious risks associated with the rural drinking water under small-central water supply mode. This one-year survey of the spatiotemporal distribution of OPs and microbial community provided scientific insights into microbial safety of rural drinking water, prompting attention on small-central water supply mode against OPs risks. [Display omitted] • A one-year survey of OP distribution in rural tap water was conducted by qPCR. • OP abundance and microbial community varied with water supply mode and season. • OP gene copy numbers in rural drinking water were higher than those in urban area. • Infectious risk under small-central water supply system needs more attention. [ABSTRACT FROM AUTHOR]