Many macrophyte species exhibit a high degree of plasticity, enabling them to thrive in various aquatic ecosystems. Identifying the growth conditions of individual aquatic plant species during research or specimen collection is not always possible. In many cases, the nature of the planned research does not necessitate recognizing environmental conditions. However, the scope of identifying the habitat parameters of the collections of submerged aquatic plant herbariums provides an opportunity for further research. This paper explores the possibilities of using isotopic signals of plants, supported by spectral analyses of powdered plant materials, to ascertain the environmental conditions from which the samples were collected. The results obtained from the stable carbon and nitrogen isotope compositions (δ13CORG and δ15NORG) and the analysis of spectral spectra via FTIR-ART (Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance) of plant material (Elodea canadensis Michx. species) collected from various habitat ecosystems, including rivers and both hard- and softwater lakes, exhibited significant distinctions between these habitats. Particularly high values of δ15NORG were recorded in the material from rivers. The stable carbon and nitrogen isotope compositions did not differentiate between the material collected from softwater and hardwater lakes. Nevertheless, when comparing the isotopic findings with the FTIR-ATR spectral analysis focused on identifying characteristic peaks associated with the presence of calcium carbonate, noticeable differences were observed in the presence and intensity of calcium carbonate peaks in the material. These differences were only evident when nondecarbonated plant material from hardwater lakes was used for the FTIR-ATR analysis. To the best of the author’s knowledge, the combination of methods applied in this study to identify the origin of E. canadensis from various freshwater environments is the first application of its kind that could enable the rapid identification of plant material origin. Such identification could prove useful in environmental, ecological, and paleoenvironmental research. The increased knowledge of macrophytes’ δ13CORG and δ15NORG values might also be essential in further tracking accelerated eutrophication based on aquatic vegetation’s isotopic signals. This might be important due to the assumption that the increased rate of eutrophication influences organic matter sedimentation in aquatic ecosystems, especially lakes.