Limestone-based freshwater ecosystems, such as the greater Everglades in South Florida, are characterized by CaCO3-rich periphyton mats that are abundant but sensitive to anthropogenic phosphorus (P) enrichment. Persistent above-ambient P exposure has been linked to mat degradation, shifts in diatom assemblages, and cascading consequences impairing ecosystem states. However, previous research shows that pH significantly influences diatom assemblages and is indistinguishable from P as a primary driver, underscoring the need for experimental studies to disentangle pH effects on mat breakdown and diatom responses to P. Here, we aim to determine how ambient pH influences the phosphorus threshold for calcareous periphyton mat breakdown and whether this response varies across the greater Everglades ecosystem. Using a 10-day ex situ mesocosm experiment, we assessed the effects of low (6.0) and high (8.0) pH on P thresholds for periphyton mat biomass, algal content, gross primary productivity (GPP), and diatom assemblage composition in samples from Everglades National Park (ENP) and Big Cypress National Preserve (BNP) using a randomized block design. The low pH treatment was associated with higher chlorophyll-a (β = 36.5, p = 0.05) and GPP (β = 1.18, p < 0.001) compared to the high pH treatment. Similarly, ENP mats showed higher chlorophyll-a (β = 90.1, p < 0.001) and GPP (β = 0.71, p = 0.001) than BNP mats. However, the P treatment had no significant effect on chlorophyll-a or GPP, and there were no significant effects of P, pH, or park identity on mat ash-free dry mass. These findings suggest low-pH, high-CO2 environments stimulate algal production and highlight differences in periphyton mat composition across the Everglades, which may influence their response to stressors. These results provide valuable insights into the interactions between pH, nutrient availability, and primary productivity that are applicable to other calcareous benthic ecosystems worldwide.