Changes in plant inputs under changing atmospheric CO2 can be expected to alter the size and/or functional characteristics of soil microbial communities which can determine whether soils are a C sink or source. Stable isotope probing was used to trace autotrophically fixed 13C into phospholipid fatty acid (PLFA) biomarkers in Mojave Desert soils planted with the desert shrub, Larrea tridentata. Seedlings were pulse-labeled with 13CO2 under ambient and elevated CO2 in controlled environmental growth chambers. The label was chased into the soil by extracting soil PLFAs after labeling at Days 0, 2, 10, 24, and 49. Eighteen of 29 PLFAs identified showed 13C enrichment relative to nonlabeled control soils. Patterns of PLFA enrichment varied temporally and were similar for various PLFAs found within a microbial functional group. Enrichment of PLFA 13C generally occurred within the first 2 days in general and fungal biomarkers, followed by increasingly greater enrichment in bacterial biomarkers as the study progressed (Gram-negative, Gram-positive, actinobacteria). While treatment CO2 level did not affect total PLFA-C concentrations, microbial functional group abundances and distribution responded to treatment CO2 level and these shifts persisted throughout the study. Specifically, ratios of bacterial-to-total PLFA-C decreased and fungal-to-bacterial PLFA-C increased under elevated CO2 compared with ambient conditions. Differences in the timing of 13C incorporation into lipid biomarkers coupled with changes in microbial functional groups indicate that microbial community characteristics in Mojave Desert soils have shifted in response to long-term exposure to increased atmospheric CO2.