Limited evidence for spatial patterns of denitrification in tidal freshwater forested wetlands (TFFWs), seemingly due to high spatial variability in the process, is surprising considering the various spatial gradients of its biogeochemical and hydrogeomorphic controls in these ecosystems. Because certain physical environmental gradients may be useful for the prediction of denitrification in TFFWs, we measured denitrification and ecosystem attributes in hummock-hollow microtopography of TFFWs along longitudinal riverine positions (upper, middle, and lower tidal river sites, and nearby upstream nontidal forested floodplains) of the adjoining Pamunkey and Mattaponi Rivers, Virginia. We tested differences by river, site, and plot in denitrification enzyme activity (DEA) and substrate limitations of denitrification potential (DP). The Pamunkey River carries greater river nitrate concentrations, and we found less nitrate limitation of DP and greater soil nitrate in hollows of this river. DEA in tidal hummocks was positively correlated with soil organic matter, nitrogen, and carbon, with the highest rates in lower tidal sites. Hummocks also promoted greater oxygen-controlled substrate limitation of DP, whereby experimental aeration stimulated DP under subsequent inundation more in hummocks than hollows. Additionally, tidal sites had greater DEA than nontidal sites, inferred to be caused by a combination of higher moisture, organic, and nutrient content. Our results indicate that the increasing nitrogen concentrations in these rivers will increase denitrification more on the Mattaponi River by alleviating its greater nitrogen limitation compared to the Pamunkey River, and modification to sedimentation, inundation, or microtopography from sea level rise may alter denitrification gradients in TFFWs and upstream low-elevation nontidal floodplains.