Microbial community structures, functional characteristics, and their relationships with hydrochemical environmental factors in arid areas of oasis area of the Hotan River Basin with fluoride-containing groundwater were investigated. Three groups of groundwater were analyzed: the LF group (ρ(F^-) ≤ 1.0mg/L), MF group (1.0mg/L<ρ (F^-) ≤ 2.0mg/L), and HF group (ρ (F^-)>2.0mg/L). The diversity, structural composition, and functional characteristics of the bacterial communities in the groundwater of the three groups were investigated using high-throughput sequencing. The results showed that ① While the diversity of the bacterial communities in the three groups was less affected by F contents, significant differences in community compositions were observed. ②Proteobacteria represented the dominant phylum in all three groups, with relative abundance of 67.61% (LF group), 52.02% (MF group), and 41.43%(HF group). The dominant bacterial genus in the HF group was Sphingobium, which had relatively low abundance in the LF and MF groups. The relative abundance of Gallionella, Hydrogenophaga, Nitrospira, and Ferritrophicum was markedly higher in the LF or MF groups compared with the HF group. ③ F^-, HCO₃^-, Ca²⁺, NO₃^-, and Cl^- were key indicators that affected bacterial community structures in F-containing groundwater. ④ PICRUSt2 functional prediction indicated that metabolic functions dominated in all three groups, while bacteria in the HF group showed reduced activities in pathways involved in co-factor abundance and vitamin metabolism compared with the LF group. ⑤ Gallionella, Hydrogenophaga, Nitrospira, and other chemoautotrophic carbon-fixing microorganisms, which had relatively high abundance in the HF group were significantly negatively correlated with the F^- and HCO₃^- contents, and significantly positively correlated with the Ca²⁺content (P<0.01). At the same time, Nitrospira was also positively correlated with NO₃^- and negatively correlated with NH₄⁺(P<0.01). The structural and functional differences in microbial communities in the groundwater of the oasis area of the Hotan River Basin, as well as their correlations with environmental factors, reflect the bidirectional relationship between chemoautotrophic carbon-fixing microorganisms and groundwater F^- contents in arid areas.