Coal mining activities produce coal mine drainage which becomes a potential pollutant of regional groundwater. The temperature and microbial community in aquifers and rock formations change with coal mining depth increases. While the influence mechanisms of temperature and microorganisms on the hydrochemical characteristics of mine water are poorly understood. This study investigated a typical minefield in north China and performed the water–rock (coal) incubation experiments to explore the effect of temperature and microorganisms on the chemical characteristics of mine water. Hydrochemistry type of coal mine water in the goafs and main water-filled aquifers of Xinjulong coal mine was the SO4–Na type. Proteobacteria and Firmicutes were the main bacterial phyla in the coal mine water. The increasing closure time of goafs decreased SO4 2− concentration and increased the microbial diversity in mine water. The 28-day incubation experiments found Na+ and SO4 2− slightly increased by 5.13 and 6.67%, respectively, and HCO3 − decreased by 14.33% when the temperature rose by 10 °C. The Shannon index of the bacterial community in water–coal system increased by 6.7% and the relative abundance of Hydrogenophaga decreased by 22.7%, while that of Thiobacillus increased by 31.56%. The microbial activities decreased Na+ and SO4 2− by 2.31 and 4.95%, respectively, but increased HCO3 − by 7.59%. The functional prediction showed the microorganisms undergone sulfur conversion reactions and affected the dissolution of ions in mine water. The results were crucial for understanding the mutual effects of temperature and microorganisms on the hydrochemical characteristics of coal mine water.