Bioclogging is a main concern in infiltration systems as it may significantly shorten the service life of these low-technology water treatment methods. In porous media, biofilms grow to clog partially or totally the pore network. Dynamics of biofilm accumulation (e.g., by attachment, detachment, advective transport in depth) and their impact on both surface and deep bioclogging are still not yet fully understood. To address this concern, a 104 day-long outdoor infiltration experiment in sand tanks was performed, using secondary treated wastewater and two grain size distributions (GSDs): a monolayer system filled with fine sand, and a bilayer one composed by a layer of coarse sand placed on top of a layer of fine sand. Biofilm dynamics as a function of GSD and depth were studied through cross-correlations and multivariate statistical analyses using different parameters from biofilm biomass and activity indices, plus hydraulic parameters measured at different depths. Bioclogging (both surface and deep) was found more significant in the monolayer fine system than in the bilayer coarse-fine one, possibly due to an early low-cohesive biofilm formation in the former, driven by lower porosity and lower fluxes; under such conditions biomass is favorably detached from the top layer, transported and accumulated in depth, so that new biomass might colonize the surface. On the other hand, in the bilayer system, fluxes are highest, and the biofilm is still in a growing phase, with low biofilm detachment capability from the top sand layer and high microbial activity in depth, resulting in low bioclogging. Overall, the bilayer coarse-fine system allows infiltrating higher volume of water per unit of surface area than the monolayer fine one, minimizing surface and deep bioclogging, and thus increasing the longevity and efficiency of infiltration systems.