The tunnel constructions were beneficial to local traffic transportation and economic development, but may cause underground water leakage and result in changes in the hydrological flow. The change in the groundwater characteristics would lead to soil micro-environmental and microbial community variations, ultimately influencing the balance of the overlying ecosystem. In this study, samples were collected from grasslands affected and unaffected by tunneling in a typical karst trough valley located at Zhongliang Mountain, Chongqing. We analyzed the soil pH, water content, and nutrient, as well as the 16S rDNA sequencing of DNA extracted from the soil. According to the comparison of the soil microbial communities between the grasslands affected and unaffected by tunneling, the dominant microorganisms at areas affected by tunneling were identified, with which the relationship between the soil pH, water content, and soil organic matter content was determined. The results showed that the total abundance and diversity of soil microorganisms, reflected by the Alpha diversity in the valley affected by tunneling, were greater than those in the valley unaffected by tunneling. The Beta diversity indicated that the microbial species in grasslands affected and unaffected by tunneling were significantly different. The Wilcoxon test identified that norank_o__iii1-15, norank_c__Gemmatimonadetes, and norank_o__MND1 were the dominant microorganisms in the grassland soils affected by tunneling and the relative abundance of the above three microbial species was more than twice of that in grassland soils unaffected by tunneling. The redundancy analysis (RDA) revealed that the soil pH and water content were the dominant environmental factors influencing the soil microbial community composition. At the areas affected by tunneling, the tunnel construction resulted in intensive groundwater leakage and a decrease in the soil water content, which led to an increase in the pH and a decrease in the soil nutrients, resulting in the increasing development of the genus norank_o__iii1-15, norank_c__Gemmatimonadetes, and norank_o__MND1, which are acclimatized to the drought-ridden, high pH, and oligotrophic soil environment in the valley affected by tunneling.