Speciation is a key determinant in the toxicity, behavior, and fate of arsenic (As) in the environment. However, little is known about the transformation of As species mediated by floating macrophytes and the phyllosphere bacteria in aquatic and wetland environment. In this study, Wolffia australiana, a rootless floating duckweed, was cultured with (W+B) or without (W-B) phyllosphere bacteria to investigate its ability in arsenite (As(III)) oxidation. Results showed that sterile W. australiana did not oxidize As(III) in the growth medium or in plant tissue, whereas W. australiana with phyllpsphere bacteria displayed substantial As(III) oxidation in the medium. Quantitative PCR of As redox-related functional genes revealed the dominance of the arsenite oxidase (aioA) gene in the phyllosphere bacterial community. These results demonstrate that the phyllosphere bacteria were responsible for the As(III) oxidation in the W+B system. The rapid oxidation of As(III) by the phyllosphere bacterial community may suppress As accumulation in plant tissues under phosphate rich conditions. The aioA gene library showed that the majority of the phyllosphere arsenite-oxidizing bacteria related either closely to unidentified bacteria found in paddy environments or distantly to known arsenite-oxidizing bacteria. Our research suggests a previously overlooked diversity of arsenite-oxidizing bacteria in the phyllosphere of aquatic macrophytes which may have a substantial impact on As biogeochemistry in water environments, warranting further exploration.