The emerging consensus on organic matter (OM) cycling in soil and sediment proposes that a continuum of biological and geochemical processes in the micro-environment controls the fate of OM. However, spatio-temporal observation of the biogeochemical nature and behaviour of OM at the soil-water interfaces (SWIs) is impeded by the heterogonous and opaque nature of their microenvironment. Herein, we used a novel SoilChip method (soil microarrays incubated with a predefined solution) to continuously mimic and trace the OM biogeochemistry at SWIs for 21 days. Combining X-ray photoelectron spectroscopy and ion sputtering on SoilChips, we provided the first direct evidence that a nanoscale organic film with a distinct composition and thickness gradually formed at the SWI within 21 days of cultivation. Although the OM coating on the SWI quickly reached equilibrium within 4 days, the formation of thicker mineral-organic association (MOA, 20-130 nm) and microbial biomass (>130 nm) continued, partially at the cost of the thin MOA (<20 nm). Consistent with the thickening SWI, the bioavailability of nutrients (dissolved organic C and ammonium) decreased gradually over 21 days, which restrained the microbial activities. Collectively, thickening SWIs act as a biogeochemical gate to regulate the bioavailability of specific organic compounds and determine their preservation or microbial mineralization. Further, thickening SWIs in thez-axis direction provide direct structural insight to increase carbon sequestration in soil and sediment. © The Royal Society of Chemistry 2020.