Understanding the spatial and seasonal dynamics of nitrogen (N)-cycle microbial communities is pivotal for the knowledge of N biogeochemistry. The present study addressed the spatial-temporal variability of nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, and nosZI) key genes, as well as of non-denitrifying nitrous oxide (N2O) reducers (nosZII), coupled with key environmental variables, in an estuarine ecosystem (Douro, NW Portugal). Samples were collected on a monthly basis over 1 yr, key physical-chemical parameters were measured, and specific functional gene abundances were assayed. The results revealed a clear seasonality for nirS, nosZII, and bacterial and archaeal amoA abundance, with an increase during the winter/spring seasons. This period was especially characterized by high levels of dissolved oxygen, low temperature, low salinity, and increased turbidity. Indeed, turbidity emerged as the key factor controlling the distribution of nirS, nosZII bacterial, and archaeal amoA abundance. In contrast, the abundance of nosZI increased during the summer, while nirK abundance was enhanced from the fall to late spring. Additionally, the availability of dissolved inorganic nitrogen nutrients had no commensurable effect on N-cycle functional genes. This study of the annual variation of N-cycle functional genes in a temperate Atlantic estuary provides a major contribution to the understanding of how environmental factors potentially influence the distribution and abundance of N-cycle microbial communities.