We integrated microscopy, spectroscopy, culturing and molecular biology, and aqueous chemistry techniques to evaluate arsenic (As) accumulation in hydroponically grown Schizachyrium scoparium inoculated with endophytic fungi. Schizachyrium scoparium grows in historically contaminated sediment in the Cheyenne River Watershed and was used for laboratory experiments with As­(V) ranging from 0 to 2.5 mg L–1 at circumneutral pH. Arsenic accumulation in regional plants has been a community concern for several decades, yet mechanisms affecting As accumulation in plants associated with endophytic fungi remain poorly understood. Colonization of roots by endophytic fungi supported better external and vascular cellular structure, increased biomass production, increased root lengths and increased P uptake, compared to noninoculated plants (p value <0.05). After exposure to As­(V), an 80% decrease of As was detected in solution and accumulated mainly in the roots (0.82–13.44 mg kg–1) of noninoculated plants. Endophytic fungi mediated intracellular uptake into root cells and translocation of As. Electron microprobe X-ray mapping analyses detected Ca–P and Mg–P minerals with As on the root surface of exposed plants, suggesting that these minerals could lead to As adsorption on the root surface through surface complexation or coprecipitation. Our findings provide new insights regarding biological and physical–chemical processes affecting As accumulation in plants for risk assessment applications and bioremediation strategies.