The capillary fringe (CF) exhibits steep chemical and redox gradients, representing a critical reactive interface in the subsurface. The spatial extent of the CF is temporally dynamic because of water table and soil moisture fluctuations. To simulate the spatio-temporal responses of the CF’s microbial community structure and activity, a 91 cm long sandy soil column experiment was conducted by imposing three different sequential hydrologic regimes: a stable water table separating the unsaturated and saturated zones (static regime), periodic pulse infiltration events with a constant water table position (infiltration regime), and water table fluctuations under three drainage-imbibition cycles of 6, 12, and 18 days (fluctuating regime). The investigation focused on the microbially-mediated turnover of nitrogen (N) and carbon (C). Geochemical profiles in the soil column were monitored continuously, while microbial DNA was extracted for 16S rDNA sequencing at the start, middle and end of the experiment. The observed microbial community compositions corresponded to five categories: (1) initial soil, (2) near-surface and (3) CF and saturated soil under the infiltration regime, (4) unsaturated and (5) saturated soil in the fluctuating regime. Based on co-occurrence network analysis, microbial physiologies matched the predominant geochemical conditions, and relatively stable community structures prevailed throughout the experiment. NO3- and NH4+ were more available to the microbes in the fluctuating regime’s alternating oxic/anoxic zone, leading to a more flexible network. The prevalent microbial metabolic pathways predicted using FAPROTAX were mainly associated with N and C metabolisms and significantly correlated with the taxonomic compositions. However, the dominant microbial groups were shared among all the soil samples. The limited spatial differences in microbial community structure under the fluctuating regime were likely due to a more pronounced role of microbial activity than microbial diversity, and similar drying-wetting conditions between the soil column and the filed site. Internally consistent geochemical and microbial depth stratifications were identified by (1) the redox potential and nitrogen species distributions, (2) spatial moment analysis of NO3- and NH4+ concentrations, (3) microbial community composition and (4) function. The five imposed hydrologic regimes generated distinct and coupled geochemical and microbial signatures. These signatures resulted from variations in the soil microbial community’s activity, rather than diversity or abundance.