The pervious lateral bars (parafluvial zone) and beds (hyporheic zone), where stream water and groundwater exchange, are dynamic sites of hydrological and biological retention. The significance of these biogeochemical 'hotspots' to stream and groundwater metabolism is largely controlled by filtration capacity, defined as the extent to which subsurface flowpaths and matrix hydraulic conductivity modify water characteristics. Where hydraulic conductivity is high, gradients in biogeochemistry and microbial activity along subsurface flowpaths were hypothesized to be less marked than where hydraulic conductivity is low. This hypothesis was tested in two riffles and gravel bars in an Australian subtropical stream. At one site, gradients in chemical and microbial variables along flowpaths were associated with reduced hydraulic conductivity, longer water residence time and reduced filtration capacity compared with the second site where filtration capacity was greater and longitudinal biogeochemical trends were dampened. These results imply that factors affecting the sediment matrix in this subtropical stream can alter filtration capacity, interstitial microbial activity and biogeochemical gradients along subsurface flowpaths. This hydroecological approach also indicates potential for a simple field technique to estimate filtration capacity and predict the prevailing hyporheic gradients in microbial activity and biogeochemical processing efficiency, with significant implications for stream ecosystem function.