The goal of this study was to provide references for the environmental management of the Three Gorges Reservoir. To study the effect of different land use patterns on soil microorganism biodiversity in the hydro-fluctuation zone of the Three Gorges Reservoir. Surface soil samples were collected from the woodland, abandoned farmland, and farmland in June 2015 to determine the bacterial and fungal community biodiversity using the terminal-restriction fragment length polymorphism (T-RFLP) and soil chemical properties in the hydro-fluctuation zone of the Ruxi River basin, in Zhongxian County, Chongqing. Soil chemical properties were also investigated. Results showed that (1) the land use patterns significantly affected soil physicochemical properties, excluding bulk weight and density. Other soil physicochemical properties, such as soil moisture, content of soil organic matter, total nitrogen, total phosphorus, available nitrogen, available potassium, and available phosphorus, in the woodland were significantly higher than those of the other two types of land use (P < 0.05); (2) there were significant differences among land use patterns in bacterial and fungal biodiversity; (3) the bacterial diversity index of the woodland was not significantly different from farmland, whereas both of them were significantly higher than abandoned farmland. Redundancy analysis and the Monte-Carlo test revealed that total phosphorus and available phosphorus showed a critical influence on bacterial diversity (P < 0.05); and (4) the Shannon-Wiener index and the Simpson index of fungal community for the woodland and abandoned farmland were significantly higher than those of farmland, whereas there was not significant difference in richness index among the three types. Redundancy analysis and the Monte-Carlo test revealed that pH, organic matter, and total nitrogen played a critical roles in influencing fungal community diversity (P < 0.05). Conclusion: compared to farmland and abandoned farmland, woodland can retain more soil nutrients, and improve soil bacterial, and fungal biodiversity, suggesting that the artificially regenerated woodland is the most appropriate land use type in the altitude of 165 m to 175 m in the hydro-fluctuation zone of the Three Gorges Reservoir. Those studies also propose that farming should be limited and the construction of artificially regenerated woodland in the hydro-fluctuation zone of the Three Gorges Reservoir region should be promoted.