Arsenic (As) is one of the most important water pollutant of global concern due to its extreme hazard. In the present study, B. subtilis synthesized iron oxide nanoparticles (Fe3O4 NPs) were used for mitigation of harmful metalloid As from the aqueous solution. Initially, the arsenic removal efficiency was tested in a batch culture experiment across various concentrations (5, 10 and 15 ppm) of B. subtilis synthesized Fe3O4 NPs at different pH, time interval and agitation speed. Optimal removal efficiency of As by using B. subtilis synthesized Fe3O4 NPs was observed at pH 7, after 80 min, and with agitation at 200 rpm. Additionally, hydroponic culture experiment was designed to assess B. subtilis synthesized Fe3O4 NPs efficiency in removal of As from As-contaminated water used to irrigate rice plants. Results revealed that B. subtilis synthesized Fe3O4 NPs effectively removed As from the contiminated water and reduced its uptake by the different parts of rice plants (root, shoot and leaf). Furthermore, these B. subtilis synthesized Fe3O4 NPs also reduced the bioaccumulation and enhanced plant tolerance to As, suggesting their potential in mitigating heavy metal toxicity, especially As and promoting plant growth. Thus, this study proposes B. subtilis synthesized Fe3O4 NPs as nano-adsorbents in reducing arsenic toxicity in rice plants.