Chemical biomonitoring (i.e., monitoring contamination in biota) is often proposed as a relevant approach to characterize the contamination of aquatic systems. Indeed, biota is an integrating matrix that enables measurements of hydrophobic substances, representative of the bioavailable fraction. Active chemical biomonitoring (i.e., using transplanted organisms from a reference site) allows controlling several parameters such as variability in exposure time, age and size of sampled organisms, therefore providing comparable results. Such active approaches are well developed in the marine environment (e.g., the French “Rinbio” network) but they are still an emerging issue for continental waters. Hence, we investigated the suitability of an active biomonitoring approach to assess trends of metals and hydrophobic organic substances contamination in continental waters. The amphipod Gammarus fossarum was selected as the test organism as i) it is a highly ecologically relevant species and ii) it allows controlling easily several biotic factors. Gammarids, sampled from a known unpolluted site, were translocated into cages to 27 sites, in the Rhône-Alpes basin (France) during early autumn 2009. Study sites represented different physico-chemical characteristics and various anthropic pressures. To avoid the influence of biotic factors, only male gammarids with the same average body length were exposed, and gammarids were fed during the experiment. After one week of exposure, concentrations of 5 metals and 28 hydrophobic organic substances, chosen with reference to the Water Framework Directive (WFD), were measured in the organisms. G. fossarum accumulated most of the investigated substances and the proposed caging methodology is a relatively simple tool for chemical biomonitoring of continental waters. To improve the discrimination between anthropically impacted and non-impacted sites, we investigated a statistical approach and a model fit to define threshold values of contamination in gammarids, assuming that any concentration under such a threshold would be assumed as the “background” concentration.