We conducted a seasonal research of the activities of microbial communities in a coastal, sandy aquifer system located in the Doñana Natural Protected Area (SW, Spain). Groundwaters from 30 piezometers were sampled over a two-year period. The proportion of active microbial biomass ranged from 0.02 to 6.36% of the total microbial biomass, while the active microbial biomass ranged from 7.42 × 10 −3 ± 2.20 × 10 −4 to 17.30 ± 3.71 ngC mL −1 . Bacterial carbon production, measured through the incorporation of [ 3 H]leucine into cellular proteins, showed a mean value of 0.18 ± 0.72 ngC mL −1 h −1 in all wells and all seasons. Bacterial growth rates ranged from 0.03 to 87.26 days. These activities exhibited spatiotemporal patterns. Temperature and the presence of nutrients and organic matter appear to be important factors controlling these patterns. However, hydrogeological flows, both local and regional, seemed to constitute the most important factor determining these spatiotemporal patterns, probably because the distribution of nutrients in aquifer systems is mainly controlled by these hydrogeological flows. The well-known hydrological flows connecting surface waters and groundwaters in Doñana support the assumption that both water compartments form a unique entity (called hydroecosystem), which functions as a whole. Consequently, not only microbial processes in surface waters can influence ecological processes in groundwaters, the characteristics of surface waters can also be affected by groundwater chemical processes, among others, mediated by the activities of microbial communities.