<p>Acid mine drainage (AMD) poses significant challenges, with arsenopyrite being a key sulfide mineral contributing to this issue. Microorganisms significantly influence arsenic behavior during arsenopyrite oxidation, yet there is limited analysis on the biooxidation process during the community coalescence of iron- and sulfur-oxidizing microorganisms. This study investigated the effects of changing the inoculation order of these microorganisms on arsenopyrite biooxidation. Geochemical analyses were used to assess solution physicochemical properties, while X-ray diffraction and X-ray photoelectron spectroscopy were used to examine the mineral phase composition. Additionally, microbial community structure and key species identification were performed using 16&#xa0;S rDNA and metagenomic analyses. Results indicated that the order of achieving biooxidation steady-state was: preferential inoculation of sulfur oxidizers &gt; preferential inoculation of iron oxidizers, reaching steady-state at 9, and 11 d, respectively. When sulfur oxidizers were preferentially inoculated, the relative content of S⁰ on arsenopyrite decreased from 17.87 to 7.89% over 5 to 18 d and the concentration of Fe³⁺ and SO₄²⁻ at 18 d was 74,037 and 237,725&#xa0;mg/kg, respectively, which were significantly higher than other groups. In all biotic groups, <i>Acidithiobacillus. ferrooxidans</i> constituted over 89% at 18 days, while <i>Acidithiobacillus. thiooxidans</i> ranged from 2.12 to 9%. This suggested that when sulfur oxidizers were preferentially inoculated, the biooxidation rate of arsenopyrite was enhanced compared to when iron oxidizers were preferentially introduced. Additionally, it also promoted the oxidation of a greater quantity of both Fe and S. This provided new perspectives on optimizing arsenopyrite treatment methods.</p>