Nitrogen (N) and phosphorus (P) concentrations and N:P ratios critically influence periphyton productivity and nutrient cycling in aquatic ecosystems. In coastal wetlands, variations in hydrology and water source (fresh or marine) influence nutrient availability, but short-term effects of drying and rewetting and long-term effects of nutrient exposure on periphyton nutrient retention are uncertain. An outdoor microcosm experiment simulated short-term exposure to variation in drying-rewetting frequency on periphyton mat nutrient retention. A 13-year dataset from freshwater marshes of the Florida Everglades was examined for the effect of long-term proximity to different N and P sources on mat-forming periphyton nutrient standing stocks and stoichiometry. Field sites were selected from one drainage with shorter hydroperiod and higher connectivity to freshwater anthropogenic nutrient supplies (Taylor Slough/Panhandle, TS/Ph) and another drainage with longer hydroperiod and higher connectivity to marine nutrient supplies (Shark River Slough, SRS). Total P, but not total N, increased in periphyton mats exposed to both low and high drying-rewetting frequency with respect to the control mats in our experimental microcosm. In SRS, N:P ratios slightly decreased downstream due to marine nutrient supplies, while TS/Ph increased. Mats exposed to short-term drying-rewetting had higher nutrient retention, similar to nutrient standing stocks from long-term field data. Periphyton mat microbial communities may undergo community shifts upon drying-rewetting and chronic exposure to nutrient loads. Additional work on microbial species composition may further explain how periphyton communities interact with drying-rewetting dynamics to influence nutrient cycling and retention in wetlands.