A population of arsenite-oxidizing microorganisms enriched from the tailing of the Shimen realgar mine was used to generate biofilms on the surfaces of perlites. This bioreactor is able to completely oxidize 1100 �gg/L As(III) dissolved in simulated groundwater into As(V) within 10 min; after 140 days of operation, approximately 20 min were required to completely oxidize the same concentration of As(III). Analysis for the 16S rRNA genes of the microbial community showed that Bacteroidetes and Proteobacteria are dominant in the reactor. Six different bacterial strains were randomly isolated from the reactor. Function and gene analysis indicated that all the isolates possess arsenite-oxidizing activity, and five of them are chemoautotrophic. Further analysis showed that a large diversity of AioAs and two types of RuBisCOs are present in the microbial community. This suggests that many chemoautotrophic arsenite-oxidizing microorganisms were responsible for quick oxidation of arsenite in the reactor. We also found that the reactor is easily regenerated and its number is readily expanded. To the best of our knowledge, the arsenite-oxidizing efficiency, which was expressed as the minimum time for complete oxidization of a certain concentration of As(III) under a single operation, of this bioreactor is the highest among the described bioreactors; it is also the most stable, economic and environment-friendly.