Physical heterogeneity determines interstitial fluxes in porous media. Nutrients and organic matter distribution in depth influence physicochemical and microbial processes occurring in subsurface. Columns 50 cm long were filled with sterile silica sand following five different setups combining fine and coarse sands or a mixture of both mimicking potential water treatment barriers. Water was supplied continuously to all columns during 33 days. Hydraulic conductivity, nutrients and organic matter, biofilm biomass, and activity were analyzed in order to study the effect of spatial grain size heterogeneity on physicochemical and microbial processes and their mutual interaction. Coarse sediments showed higher biomass and activity in deeper areas compared to the others; however, they resulted in incomplete denitrification, large proportion of dead bacteria in depth, and low functional diversity. Treatments with fine sediment in the upper 20 cm of the columns showed high phosphorus retention. However, low hydraulic conductivity values reported in these sediments seemed to constraint biofilm activity and biomass. On the other hand, sudden transition from coarse-to-fine grain sizes promoted a hot-spot of organic matter degradation and biomass growth at the interface. Our results reinforce the idea that grain-size disposition in subsurface sandy sediments drives the interstitial fluxes, influencing microbial processes.