Alnus spp. are actinorhizal trees commonly found in wet habitats and able to grow effectively in soil slightly contaminated with metal trace- elements. Two clones belonging to two Alnus species, namely, A. incana and A. glutinosa, were grown in hydroponics and exposed for 9 weeks to a Cd + Ni + Zn polymetallic constraint. Although responding by a similar decrease in total biomass production, the proteomic analysis associated with the study of various biochemical parameters including carbohydrate and mineral analyses revealed that the two clones have a distinct stress-responsive behavior. All parameters indicated that the roots, the organ in direct contact with the media, are more affected than the leaves. In fact, in A. glutinosa the response was almost completely confined to the roots, whereas many proteins change significantly in the roots and in the leaves of the treated A. incana. In both clones, the changes affected a broad range of metabolic processes such as redox regulation and energy metabolism and induced the production of pathogenesis-related proteins. In particular, changes in the accumulation of bacterial proteins that were not identified as coming from the known symbionts of Alnus were reported. Further investigation should be performed to identify their origin and exact role in the plant response to the polymetallic exposure tested here.