Pollutant loads stemming from anthropogenic activities conveyed in urban stormwater runoff contribute to the impairment of downstream water bodies. Cities and municipalities are increasingly turning toward green infrastructure stormwater control measures to treat pollutants at the source of runoff. One example of these technologies is bioretention, which is commonly applied for stormwater treatment in urban areas due to its demonstrated effectiveness in removing various pollutants from water, including sediment, nutrients (e.g., N and P), and metals. As metals are mainly removed by filtration or adsorption to soil particles, the filter media is important for metal removal in bioretention. However, the capacity to remove metals through adsorption by bioretention media is finite; thus, the media may need to be replaced and disposed of after maintenance or at the end of its operational lifespan. Pollutant accumulation in bioretention media has the potential to approach toxicity thresholds, which may introduce complexities for safe handling and disposal. To fully capture the potential challenges associated with metals accumulation in media over time, it is important to understand the accumulation processes and mobility of metals in bioretention facilities as they age. Although several studies have investigated metal accumulation and distribution in bioretention media, few have assessed metal mobility by fractionation using sequential extraction methods in older (i.e., >7 years) facilities. In November 2019, we conducted a comprehensive field study of older facilities in Ohio, Michigan, and Kentucky (USA) to improve the understanding of the accumulation processes and metal mobility in bioretention. In this study, concentrations of several metals (i.e., Cr, Cu, Ni, Pb, and Zn) were analyzed in samples of filter material from 29 bioretention sites in operation for 7–16 years. Except for Cd, all metals were found in all samples. Metals accumulation was clear with highest concentrations found in the top (0–5 cm) layer of the filter material, attributable to the filtration of particles percolating through the media profile. Lower concentrations were observed in deeper (i.e., >10 cm) layers of the bioretention media. The fractionation showed that the metals of interest were present at high levels with a risk of leaching over time, among which Cd, Zn, and Pb were suggested to be mobile from the filter material during precipitation. Thus, there is a potential risk of leakage from filter material or sediment removed from biofilters, e.g., during maintenance and disposal. The results of principal component analysis indicated specifically correlations between metal concentrations and the filter material soil texture including the organic matter content. These results contribute to improved design and operation and suggest regular maintenance to reduce long-term risks associated with the accumulation of metals in bioretention and similar urban stormwater treatment facilities. Since most metals are trapped in the top layer of the filter it may be enough to remove only the top layer. However, metal fractionation should be considered when handling the material.