Subsurface flow pathways of groundwater-fed streams were characterized on a floodplain terrace of the Toklat River, Alaska, in summer 2008, to establish the influence of local physicochemical variability upon macroinvertebrate communities. Streams proximal to the valley side (A sites) and to the main meltwater channel (B sites) were studied. Chloride and natural isotopic tracers (δ¹⁸O and δ²H) were used to identify water sources and flow pathways. Results indicated that flow in B sites was dominated by seepage of glacial meltwater through the alluvial aquifer. Streamflow at sites situated at higher elevations was ephemeral, and commenced with the seasonal rise in the groundwater table. In contrast, the physicochemistry of A sites was characteristic of seepage from valley-side debris fans, which maintained perennial flow to streams at lower elevations. Macroinvertebrate diversity was lower in ephemeral streams, likely due to colonization constraints. In June macroinvertebrate abundance was significantly positively correlated with the percentage contribution to streamflow from debris-fan seepage (p < 0.05) and with fine particulate organic matter concentration (FPOM) (p < 0.05); FPOM was correlated with debris fan seepage (p < 0.05). These relationships were not evident in July and August, when organic matter availability increased. Our study demonstrates that flow pathways and organic matter availability significantly influence macroinvertebrate communities in these groundwater-fed streams.