To better understand how water stress and availability affect the structure of microbial communities in soil, I measured the change in phospholipid fatty acids (PLFA) and the incorporation of 13C-labeled glucose into the PLFA following exposure to water stress. Overlaid on the laboratory water stress treatment, samples were collected from drought-prone and irrigated (11 years) tallgrass prairie soil (0–10 cm depth). In the laboratory, soils were either incubated at −250 kPa or dried steadily over a 3-d period to −45 MPa. On the fourth day, the dried samples were brought up to −250 kPa and then all samples received 250 μg of glucose-C (+4000 δ13C-PDB) solution that brought them to −33 kPa matric water potential. Samples were then extracted for PLFA following 6 and 24 h of incubation (25 °C). Non-metric multidimensional scaling (NMS) techniques and multi-response permutation procedure (MRPP) showed that the largest effect on the mol% distribution of PLFA was related to the field scale water addition experiment. In response to irrigation, the PLFA 16:1ω5, 18:1+, and 18:2ω6,9 showed increases, and a15:0, a17:0, and cy19:0 showed decreases in their respective mol%. Effects related to the induction of laboratory water stress were predominantly associated with a decrease in the mol% distribution of the putative fungal biomarker (18:2ω6,9) with little to no change in the mol% distribution of the bacterial biomarkers. Interestingly, the flow of C to the microbial community was not strongly related to any single PLFA, and differences were rather subtle, but multivariate MRPP detected change to the community structure related to the laboratory water stress treatment but not related to the 11 years of field irrigation. Our results suggest that both the total and the actively metabolizing bacterial community in soil were generally resistant to the effects of water stress brought by rewetting of dry soil. However, more research is needed to understand the nature of the fungal response to drying and rewetting in soil.