Given the increasing need to protect vulnerable freshwater ecosystems and make them more resilient to human use and climate change, biomonitoring of the hyporheic zone (HZ), which plays a critical role in pollution attenuation, is essential. The aim of the present study was to assess the potential of the amphipod species Synurella ambulans as a bioindicator of metal contamination in the HZ of the Sava River (Croatia). Amphipods were collected during the four seasons at two sampling sites (average sampling depth 55 cm) differing in type (agricultural and urban) and intensity (diffuse and point source contamination) of anthropogenic influence, one located upstream (Medsave), and the other downstream (Jarun) of the wastewater treatment plant discharge. Concentrations of Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Rb, Sn, Zn, Ca, K, Mg and Na were measured in the interstitial water, sediments and specimens of S. ambulans by HR ICP-MS. Physicochemical parameters (temperature, DO, O₂ saturation, pH, conductivity, alkalinity, total water hardness, COD_KMnO4, nutrients) were measured in the interstitial water, while organic carbon was measured in the interstitial water and sediments. Metal concentrations in interstitial water and sediments were below thresholds set by environmental quality standards. Metal concentrations in S. ambulans were classified as follows: higher at the Jarun site (Al, Cr, Fe, Ni, Pb, Sn), higher at the Medsave site (Cd, Cu, Rb) and mostly comparable at both sites (Co, Mn, Zn). Bioaccumulation factors were generally higher at Jarun, with average values ranging from 322 to 143,278 L kg⁻¹. Bioaccumulation of metals in S. ambulans depended on various environmental factors, with metal exposure level and dissolved macro elements showing the strongest association with metals accumulated in S. ambulans. The findings provided the first evidence on the suitability of S. ambulans as a good bioindicator of chronic metal contamination in the HZ.