Understanding and predicting the extent, location, and function of biogeochemical hot spots at the watershed scale is a frontier in environmental science. We applied a hydropedologic approach to identify (1) biogeochemical differences among morphologically distinct hydropedologic settings and (2) hot spots of microbial carbon (C) and nitrogen (N) cycling activity in a northern hardwood forest in Hubbard Brook Experimental Forest, New Hampshire, USA. We assessed variables related to C and N cycling in spodic hydropedologic settings (typical podzols, bimodal podzols, and Bh podzols) and groundwater seeps during August 2010. We found that soil horizons (Oi/Oe, Oa/A, and B) differed significantly for most variables. B horizons (>10 cm) accounted for 71% (±11%) of C pools and 62% (±10%) of microbial biomass C in the sampled soil profile, whereas the surface horizons (Oi/Oe and Oa/A; 0–10 cm) were dominant zones for N-cycle-related variables. Watershed-wide estimates of C and N cycling were higher by 34 to 43% (±17–19%) when rates, horizon thickness, and areal extent of each hydropedologic setting were incorporated, versus conventionally calculated estimates for typical podzols that included only the top 10 cm of mineral soil. Despite the variation in profile development in typical, bimodal, and Bh podzols, we did not detect significant differences in C and N cycling among them. Across all soil horizons and hydropedologic settings, we found strong links between biogeochemical cycling and soil C, suggesting that the accumulation of C in soils may be a robust indicator of microbial C and N cycling capacity in the landscape.