Forest plants that can assimilate nitrate may act as nitrate sink and, consequently, reduce nitrate losses from watershed ecosystems through leaching. This study, conducted at the Fernow Experimental Forest in West Virginia, quantified via nitrogen reductase activity (NRA) the nitrate assimilation of two tree species, red maple and sugar maple, and surrounding common herb-layer species at the tissue (foliage, roots) and plot level. NRA measurements were conducted in summer and spring. Furthermore, NRA was quantified under varying levels of soil nitrate availability due to fertilization, different stages in secondary forest succession, and watershed aspect. This study confirmed that NRA of mature maples does not respond to varying levels of soil nitrate availability. However, some herb-layer species’ NRA did increase with nitrogen fertilization, and it may be greater in spring than in summer. Combined with biomass, the herb layer’s NRA at the plot-level (NRAA) comprised 9 to 41% of the total (tree + herb-layer) foliar NRAA during the growing season. This demonstrates that the herb layer contributes to nitrate assimilation disproportionally to its small biomass in the forest and may provide a vernal dam to nitrate loss not only by its early presence but also by increased spring NRA relative to summer.