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Northeast Shark River Slough (NESS), lying at the northeastern perimeter of Everglades National Park (ENP), Florida, USA, has been subjected to years of hydrologic modifications. Construction of the Tamiami Trail (US 41) in 1928 connected the east and west coasts of SE Florida and essentially created a hydrological barrier to southern sheet flow into ENP. Recently, a series of bridges were constructed to elevate a portion of Tamiami Trail, allow more water to flow under the bridges, and attempt to restore the ecological balance in the NESS and ENP. This project was conducted to determine aspects of soil physiochemistry and microbial dynamics in the NESS. We evaluated microbial respiration and enzyme assays as indicators of nutrient dynamics in NESS soils. Soil cores were collected from sites at certain distances from the inflow (near canal, NC (0–150 m); midway, M (150–600 m); and far from canal, FC (600–1200 m)). Soil slurries were incubated and assayed for CO2 emission and β-glucoside (MUFC) or phosphatase (MUFP) activity in concert with physicochemical analysis. Significantly higher TP contents at NC (2.45 times) and M (1.52 times) sites than FC sites indicated an uneven P distribution downstream from the source canal. The highest soil organic matter content (84%) contents were observed at M sites, which was due to higher vegetation biomass observed at those sites. Consequently, CO2 efflux was greater at M sites (average 2.72 µmoles g dw−1 h−1) than the other two sites. We also found that amendments of glucose increased CO2 efflux from all soils, whereas the addition of phosphorus did not. The results indicate that microbial respiration downstream of inflows in the NESS is not limited by P, but more so by the availability of labile C.