The Peace River is a critical water source in southwest Florida, United States. The watershed contains many phosphate mines that decrease water safety. Whether phosphate mining leads to a reduction in surface runoff and affects water quality in the Peace River Basin has been a highly controversial subject. Thus, the environmental impacts of phosphate mining in the Peace River were assessed. The Soil and Water Assessment Tool (SWAT) model is a widely used physical-mechanism-based distributed hydrological model that uses spatial distribution data, such as topography, soil, land use, and weather, to predict water, sediment, nutrient, pesticide, and fecal bacteria production. Based on a SWAT model, runoff, total nitrogen (TN) load, and total phosphorus (TP) load at the outlet of the Peace River Basin from 2001 to 2018 were investigated. The applicability of the four uncertainty methods in the hydrological simulation of the basin was assessed. The runoff at five stations in a specified mining area was simulated to analyze the impact of human-dominated land use changes caused by phosphate mining on the water environment. The results for the pre- and post-mining periods showed that the land use transfer in the study area experienced large fluctuations and that the land use change had a significant impact on the runoff (the outlet site decreased by 44.14%), indicating that phosphate mining has a significant effect on reducing runoff in the basin. An analysis of three scenarios (pre-mining [s1], post-mining [s2], and reclamation [s3]) showed that during s1–s2–s3, the change in mining land area is large (increased by 142.86%) and that TN and TP loads increased, indicating that human activities mainly affect the water environment through phosphate mining. This is mainly because a large amount of wastewater containing high concentrations of inorganic chemicals, which is produced in the process of phosphate mining and processing, overflows directly or from the sedimentation tank into the river. In summary, the simulation results showed that the changes in runoff and pollutants were attributed to phosphate rock mining. Therefore, strengthening the management of phosphate mining and adopting effective protection measures is of substantial significance for the effective protection of water resources. By analyzing the measured data, this study can help people understand more actual situations and further evaluate the impact of phosphate mining activities on the water environment. The simulation results can also be used to predict the future trend of runoff and water quality in the Peace River Basin and provide a decision-making basis for government management departments to issue water resource protection measures.