Paddy soil experiences repeated anaerobic and aerobic changes during rice growth, the dramatic dynamics of soil water status accompanied by changes in redox condition and O₂ availability. However, the effect of rapid water status change on soil microbial biomass and community composition is not well explored. Here, we present a comprehensive study focusing on the short-term water status change in 13 Chinese paddy soils. In order to gain a reliable way to determine soil microbial biomass carbon (MBC) in flooded or water-saturated soils, we also evaluated two different procedures (nitrogen bubbled and 100000200°C water bath) to remove chloroform in extracts during the fumigation process. Compared to non-flooded paddy soils, the flooded paddy soils tended to have a lower microbial biomass, and this was much clearer using adenosine 5'-triphosphate (ATP) and phospholipid fatty acid (PLFA) analysis compared to biomass measured by the fumigation method. Fungal biomass, which was indicated by both ergosterol and the PLFA 18:203096,9c, also decreased after short-term flooding. Changes in soil microbial community composition (determined by PLFA biomarkers) were observed after short-term flooding, but the extent varied between soils. This study indicates that the dynamics of short-term water status altered the soil microbial biomass (ATP, MBC, and total PLFA) and community composition. Finally, our results suggested that liquid fumigation combined with the nitrogen-bubbled method is the best choice for analyzing MBC concentrations in water-saturated soils.