Abstract Flooding affects both above- and below-ground ecosystem processes. While the below-ground changes may be less obvious, they are as important as the above-ground changes. Soil microorganisms are sensitive to disturbance, and shifts in soil microbial community structure are expected when anaerobic conditions develop from flooding. The primary objective of these studies was to determine the effect of flooding on soil microbial communities. Simulated floods were established under greenhouse and field conditions. Flood treatments of flowing, intermittent (greenhouse only) or stagnant conditions were compared to a control with no flooding. In addition, residue treatments (incorporation of grass, legume or tree residue) were evaluated under greenhouse conditions. Soil samples from these experiments were examined using phospholipid fatty acid (PLFA) analysis and total N (TN), total organic carbon (TOC) and C:N ratio. Stagnant flood conditions in the greenhouse decreased microbial biomass and markers for aerobic bacteria, Gram-negative bacteria, Gram-positive bacteria, and mycorrhizal fungi. However, residue treatment, in general, did not affect microbial community structure. Effects of flood treatments in the field varied with depth and flood × depth interaction. The B:F ratio and microbial biomass decreased with stagnant flooding while other measures were not affected by flooding. Microbial biomass and microbial markers decreased with depth. We found some changes in the soil microbial community due to flooding; however, the 5-week time period of our study may not have been long enough to develop measureable changes. Further changes in the microbial community may occur as flood waters remain in a given area.