In this study, the anaerobic quartz sand fixed biofilm reactor containing Cupriavidus sp. W12 was established to simultaneously remove calcium (Ca2+), fluoride (F−) and nitrate (NO3–N) from groundwater. After 84 days of continuous operation, the optimum operating parameters and defluoridation mechanism were explored, and the microbial community structure under different pH environments were compared and analyzed. Under the optimal operation conditions (HRT of 6 h, initial Ca2+ concentration of 180 mg L−1, and pH of 7.0), the removal efficiencies of Ca2+, F−, and NO3–N were 58.97%, 91.93%, and 100%, respectively. Gas chromatography (GC) results indicate that N2 is the main gas produced by the bioreactor. Three−dimension excitation emission matrix fluorescence spectroscopy (3D−EEM) showed that extracellular polymers (EPS) are produced during bacterial growth and metabolism. The results of Scanning electron microscopy−energy dispersive spectrometer (SEM−EDS), X−ray diffraction (XRD), and Fourier transform infrared spectrometer (FTIR) demonstrated that the defluoridation mechanism is attributed to the synergetic effects of ion exchange, co−precipitation, and chemisorption. The comparative analysis of the microbial community structure under different pH conditions show that Cupriavidus is the dominant bacteria in the bioreactor throughout the experiment, and it shows a prominent advantage at pH of 7.0. This research provides an application foundation for anaerobic microbial induced calcium precipitation (MICP) bioremediation of Ca2+, F−, and NO3–N from groundwater.