The phospholipid fatty acids (PLFA) as biomarkers of microbial community structures were appointed to elaborate the mobilization of arsenic (As) in groundwater from Datong Basin. The results showed that saturated fatty acids 16:0, 18:0, monounsaturated fatty acids 16:1ω9, 18:1ω9, and cyclopropane fatty acids 17:0, 19:0 had relatively high contents. This may imply the significant impact of dominant Desulfobacter and Clostridium on the mobilization of As in the shallow groundwater. Sulfate-reducing bacteria and Fe-reducing bacteria can mediate the reductive dissolution of As-bearing Fe oxides/hydroxides to cause the release of As into groundwater.