This study aimed to investigate the hydrochemical characteristics of different aquifers, controlling factors, bacterial community structural characteristics, and their interaction with groundwater environmental factors in typical coal mines in the Huaibei Coalfield. A total of nine groundwater samples from the Cenozoic Loose Layer Pore (Second and Third), Carboniferous Taiyuan Group Tuff Karst Fissure, and Ordovician Tuff Karst Fissure aquifers were collected and used for water chemistry analysis and bacterial 16S rRNA gene sequencing. The overall groundwater in the study area was weakly alkaline, and the hydrochemical type of groundwater was dominated by the SO4•Cl-Na type, followed by the SO4•Cl-Ca•Mg and HCO3-Mg•Ca types. The weathering of silicate and carbonate rocks controlled the formation of the chemical components of the groundwater, and the groundwater tended to develop from silicate to carbonate rocks. The dominant microbes in groundwater from different aquifers were Bacillaceae and Streptococcaceae, but their abundance was significantly affected by the depth; the abundance of the two bacterial groups was loose layer aquifer > Taiyuan limestone aquifer > Ordovician limestone aquifer. HCO3 - and F- were the main factors affecting the distribution of bacterial community structure. The study’s results provide theoretical references for exploring the hydrochemical formation mechanisms and microbiological properties of different aquifers, as well as potential references for utilizing the differences in bacterial properties for identifying water sources during mine water emergencies.