Algal and plant production of nonphosphorus lipids in place of phospholipids is a physiological response to low phosphorus (P) availability. This response has been shown in culture and in marine plankton studies, but examples from freshwater algae remain minimal. Herein, we analyzed the nutrient contents and lipid composition of periphyton communities across the Florida Everglades ecosystem. We hypothesized that in phosphate-poor areas, periphyton in high- and low-sulfate waters would vary the proportion of sulfolipids (SLs) and betaine lipids (BLs), respectively. In phosphate-enriched areas, periphyton would produce more phospholipids (PLs). We observed that at low-P sites, PLs were a minor lipid component. In cyanobacteria-dominated periphyton where sulfate was abundant, BLs were only slightly more abundant than SLs. However, in the low-P, low-sulfate area, periphyton were comprised to a greater degree green algae and diatoms, and BLs represented the majority of the total lipids. Even in a P-rich area, PLs were a small component of periphyton lipid profiles. Despite the phosphorus limitations of the Everglades, periphyton can develop tremendous biomass. Our results suggest a physiological response by periphyton to oligotrophic conditions whereby periphyton increase abundances of nonphosphorus lipids and have reduced proportions of PLs.