A large-scale stormwater biofilter column study was conducted to evaluate the impact of design configurations and operating conditions on metal removal for stormwater harvesting and protection of aquatic ecosystems. The following factors were tested over 8 months of operation: vegetation selection (plant species), filter media type, filter media depth, inflow volume (loading rate), and inflow pollutant concentrations. Operational time was also integrated to evaluate treatment performance over time. Vegetation and filter type were found to be significant factors for treatment of metals. A larger filter media depth resulted in increased outflow concentrations of iron, aluminum, chromium, zinc, and lead, likely due to leaching and mobilization of metals within the media. Treatment of all metals except aluminum and iron was generally satisfactory with respect to drinking water quality standards, while all metals met standards for irrigation. However, it was shown that biofilters could be optimized for removal of iron to meet the required drinking water standards. Biofilters were generally shown to be resilient to variations in operating conditions and demonstrated satisfactory removal of metals for stormwater-harvesting purposes.