Lipids were extracted in order to explore the microbial communities in eleven biofilms and four layers of two stromatolite samples, from the Green Valley acid mine drainage site in western Indiana. The high concentrations of phytadiene, polyunsaturated fatty acids, phytol, phytanol, and monounsaturated fatty acid 18:1Δ⁹ in the biofilms indicated the abundance of phototrophs, including microeukaryotes, algae (mainly diatoms), and fungi. Wax esters in the hydrocarbon fraction possibly suggested the presence of localized anaerobic zones within the biofilms, where the microeukaryotes synthesized the esters by an unusual reverse β-oxidation pathway. Other microbial communities in the biofilms mainly included sulfate-reducing bacteria (SRB), and Gram-positive bacteria. The contribution of cyanobacteria to the lipid pool was not very significant. The trans/cis ratio of monounsaturated fatty acids can be a good indicator of the physiology of the biofilms, indicating whether it is attached or floating, and further depicting the nutrient conditions in which they thrive. The primary cause of ¹³C enrichment in the GVS biomarkers has been attributed to a carbon-limiting system existing in the mine environment. The depletion of carbon is the result of increased sulfate, resulting in lowering of pH in the AMD. Both autotrophic and heterotrophic eukaryotes were detected by the biomarker study. While the autotrophs used aqueous CO₂ for carbon fixation, heterotrophs either used the autotrophic dissolved organic carbon (DOC) or C₃/C₄ organic matter for the same. A model for carbon flow has been proposed to explain the biogeochemistry in GVS.