The Everglades Stormwater Treatment Areas (STAs), important components in the Everglades Restoration efforts, were built to remove excess phosphorus (P) from stormwater runoff before it enters the Everglades Protection Area. The STAs operate within the low phosphorus domain of constructed wetlands and are mandated to achieve ultra-low P outflow concentrations to protect the receiving water bodies. Microbial processing by periphyton enzymatic activity under these low-P conditions may be critical for achieving nutrient reduction, particularly in the lower reaches of the STA treatment cells where nutrients are primarily in the dissolved organic and particulate forms. Enzymes are produced by periphyton to liberate required nitrogen (N), carbon (C), and P from dissolved organic matter under nutrient limiting conditions. Enzyme activity had not been measured within the STA treatment cells and this study was conducted to evaluate if enzyme activity occurs along the nutrient transect from inflow to outflow in well-performing treatment cells and if this activity differs within the dominant vegetation communities (i.e., emergent or submerged aquatic vegetation, EAV and SAV, respectively). The STAs receive variable hydraulic and nutrient loadings and the potential influence of these conditions on enzyme activity was evaluated under a range of loadings. The periphyton response in three different well-performing STA treatment cells (STA-2 Cells 1 and 3 and STA-3/4 Cell 3B) was evaluated using periphyton that accrued in the water column over a 7-d period. Because nutrient cycling is interrelated, a suite of enzyme activity associated with nutrient acquisition from dissolved organic material was measured. Enzymes associate with P-acquisition (phosphatase; alkaline phosphatase and bis-phosphodiesterase), C-acquisition (β-glucosidase), and N-acquisition (leucine aminopeptidase) were measured along internal transects from inflow to outflow.We found that enzyme activity occurs in the STA treatment cells and the most reactive enzymes were the phosphatase and N-acquisition enzymes; there was little activity and variability measured in the C-acquisition enzyme. Phosphatase activity increased along the nutrient gradient and the rates became more variable at the midflow and outflow transect locations. When dissolved organic P (DOP) in the surface water was above 10 μg/L, the phosphatase rates were reduced, an expected response since enzyme production occurs only during nutrient limiting conditions. When DOP concentrations were <10 μg/L, greatly increased rates were observed but there were still also times when the rates were reduced that did not appear to be influenced by surface water organic P or N concentrations or water depths. The activity for N-acquisition enzyme was also found to be variable, but this variability was between each treatment cell rather than along the transect as observed for phosphatase activity. The proportional change of each enzyme activity to the other was used to evaluate nutrient limiting conditions in the periphyton. Along the transect, both N- and P-limiting conditions were found in the periphyton at the inflow and midflow sites and solely P-limiting conditions at the outflows in all treatment cells. This implies that the mineralization processes can be disrupted under higher nutrient conditions, impacting cycling of the organic material. There were also indications that nutrient cycling may differ between periphyton from the EAV and SAV communities under low-P conditions, suggesting there may be a complementary enzymatic response within a mixed marsh configuration that may enhance nutrient cycling.