To explore the response of soil microbes to seasonal water fluctuation, soil samples were collected from three sampling belts with different elevations in the Poyang Lake wetland over four seasons (Mar, Jun, and Oct 2014, Jan 2015). Soil physicochemical properties, microbial basal respiration, microbial biomass, and enzyme activities were measured. The results showed that (1) the contents of soil organic carbon, dissolved organic carbon, and available phosphorus varied with seasonal water level fluctuations, and the seasonal dynamics of microbial biomass carbon (MBC) and four hydrolases, including β-glucosidase (Bglu), β-xylanase (Bxyl), N-acetylglucosaminidase (NAG), and phosphatase (Phos) were affected. MBC, Bglu, Bxyl, NAG, and Phos peaked in summer and winter, but decreased in autumn, whereas the two oxidases, phenoxidase (Phox) and peroxidase (Pero), showed the opposite trend; (2) apparent impacts of water level elevation and aboveground vegetation were observed, and higher nutrients and microbial activities were found in S2 plots covered with Triarrhena lutarioriparia L. Liou; (3) some soil properties (soil moisture, ammonia-nitrogen, organic carbon, available phosphorus, etc.) were significantly correlated to microbial activities (microbial biomass, basal respiration, and enzymes); and the impact of seasonal water level changes on microbial activities was greater than that of elevation differences. This study revealed that water regime shifts had an important impact on soil microbial activities, which would further affect the ecosystem function wetland.