Alterations in microbial community composition, biomass, and function in the Florida Everglades impacted by cultural eutrophication reflect a new physicochemical environment associated with monotypic stands of Typha domingensis. Phospholipid fatty acid (PLFA) biomarkers were used to quantify microbial responses in detritus and surface soils in an active management experiment in the eutrophic Everglades. Creation of open plots through removal of Typha altered the physical and chemical characteristics of the region. Mass of PLFA biomarkers increased in open plots, but magnitude of changes differed among microbial groups. Biomarkers indicative of Gram-negative bacteria and fungi were significantly greater in open plots, reflective of the improved oxic environment. Reduction in the proportion of cyclopropyl lipids and the ratio of Gram-positive to Gram-negative bacteria in open plots further suggested an altered oxygen environment and conditions for the rapid growth of Gram-negative bacteria. Changes in the PLFA composition were greater in floc relative to soils, reflective of rapid inputs of new organic matter and direct interaction with the new physicochemical environment. Created open plot microbial mass and composition were significantly different from the oligotrophic Everglades due to differences in phosphorus availability, plant community structure, and a shift to organic peat from marl-peat soils. PLFA analysis also captured the dynamic inter-annual hydrologic variability, notably in PLFA concentrations, but to a lesser degree content. Recently, use of concentration has been advocated over content in studies of soil biogeochemistry, and our results highlight the differential response of these two quantitative measures to similar pressures.