Microbial-mediated FeS oxidation to Fe(Ⅲ) minerals via chemolithoautotrophic Fe(Ⅱ) oxidizers under pH/O₂ limitations engages As immobilization. However, this process is constrained under the dual stress of micro-oxygen and acidic conditions due to the critically diminished Fe(Ⅱ) oxidation capacity. Therefore, the interplay between Fe(Ⅱ) oxidation, carbon metabolism, and As immobilization in Fe(Ⅱ)-oxidizing bacteria under micro-oxygen and acidic conditions remains unclear. This study presents the first successful enrichment of microaerophilic and acidophilic Fe(II)-oxidizing bacteria (MAFeOB). These bacteria are capable of oxidizing FeS to Fe(III) minerals and immobilizing up to 27,835 mg/kg of As(Ⅴ) under micro-oxygen content (below 3.2 mg/L) and acidic pH (4.5-6.2). Through comprehensive metagenomic analysis, it was speculated that MAFeOB harbor a suite of genes potentially participating in critical processes, including carbon fixation, Fe(II) oxidation, and arsenic detoxification. Notably, a potential electron transfer pathway from Cyc2_repCluster2 to Cytochrome cbb3-type oxidases facilitates Fe(II) oxidation. Furthermore, As(Ⅲ) efflux pump (arsA, arsB, acr3) and As(Ⅲ) oxidase (aioA) genes indicate MAFeOB's potential for As immobilization. Our findings underscore the pivotal role of MAFeOB in overcoming limitations associated with Fe(III) mineral formation, thereby enhancing arsenic immobilization under micro-oxygen and acidic water.