Eutrophication of the Florida Everglades, USA, has altered the characteristics of the ecosystem, but management strategies are being implemented to accelerate recovery. In this study, we described lipid compositional similarities and differences between periphyton, fish, and crustaceans, and explored if eutrophication and creation of new open-water sloughs in phosphorus (P)-impacted regions of a Northern Everglades impoundment resulted in changes in periphyton biomass and lipid composition, and the lipid composition of a ubiquitous omnivore, Gambusia holbrooki. Lipid biomarker analysis provided insight into microbial community composition, quality of basal resources, and potential resources utilized by consumers. Periphyton biomass and phospholipid fatty acid (PLFA) composition differed in response to eutrophication, but not between P-impacted control and treatment plots. Shifts in relative abundances of lipids indicative of diatoms and green algae mirrored known taxonomic shifts due to eutrophication. For fauna, PLFA were a small and relatively distinct component of the overall total lipid make-up, and profiles were similar between control and treatment plots. However, the PLFA profile of G. holbrooki differed between oligotrophic and eutrophic regions. Fish and crustacean lipids contained significantly greater relative abundances of polyunsaturated fatty acids than were found in periphyton, and profiles differed between fish and crustaceans, suggesting organisms were selectively accumulating or elongating and desaturating lipids de novo, to meet physiological needs. This study builds on findings of microbial responses to eutrophication and recent observations that consumer PLFA profiles can also shift with P-enrichment.