The coexistence of F - , Ca 2+ , nitrates, and other pollutants in water body has aroused widespread concern. In this research, a novel quartz sand biofilm reactor was established, aiming to study the key factors of different carbon to nitrogen (C/N) ratios (5:1, 4:1, and 3:1), initial Ca 2+ concentration (180 mg L -1 , 144 mg L -1 , and 108 mg L -1 ), and hydraulic retention time (HRT) (4 h, 6 h, and 8 h) on simultaneous Ca 2+ precipitation, F - removal, and denitrification. Results showed that the removal efficiencies of Ca 2+ , F - , and nitrate were 55.04%, 82.64%, and 97.69% under the low C/N ratio of 3:1, initial Ca 2+ concentration of 180 mg L -1 , and HRT of 8 h. 3-D Excitation-Emission Fluorescence Spectroscopy (3-D EEM) demonstrates that extracellular polymeric substances (EPS) was generated during the growth metabolism. Scanning Electron Microscopy (SEM) and X-ray diffractometer images showed that Ca 2+ , F - removed in the form of CaCO 3 , Ca 5 (PO 4 ) 3 F and CaF 2 under Acinetobacter sp. H12 induction. Moreover, high-throughput sequencing results display that the biomineralized bacteria Acinetobacter sp. H12 exerted great influence in the bioreactor. This research will underpin the practical use of multiple pollutants such as F - and Ca 2+ wastewater under the different C/N ratios., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)