Softwater lakes with specific, rare, and protected aquatic plant vegetation are very sensitive to increased trophic and water chemical changes, especially alkalization. These changes might be reflected in the stable carbon and nitrogen isotopes of the organic matter (OM) of those plants (δ13CORG and δ15NORG) and sediments (δ13CORG and δ15NTN) which they cocreated. To recognize the relationship between OM in the aquatic plants of softwater lakes and the cocreated sediments, we analyzed the plants δ13CORG and δ15NORG in light of four ecological groups (as well as the sediments δ13CORG and δ15NTN in which these plants thrived). Studies were performed in July 2020 on 14 softwater lakes in northern Poland with varying pH gradients from 4.86 to 9.20. For each lake, a single stand was examined for each species detected. The goal was to investigate each species at 10 sites, but this target was not reached for several species. Among the investigated ecological groups, isoetids and mosses showed the least variation in obtained values. In contrast, elodeids exhibited the highest variation in δ13C results due to their diverse carbon uptake strategies, involving both CO2 and HCO3− forms. Moreover, the δ13C values of charophytes and elodeids were highly related to the increasing pH of water. The slight differences between the δ13C results of plants and sediments in isoetid stands further support this finding. Furthermore, we noticed an increase in sediment δ13CORG values along the alkalization gradient, suggesting that macrophytes were the primary source of OM for the sediments. The positive correlation between the δ13C values of plants and sediments (r = 0.69, p < 0.05) might also confirm that the macrophytes were a significant source of OM in sediments. Regarding δ15N values, we did not find significant differentiation between plants and sediments across ecological groups. However, sediment consistently exhibited 15N-enrichment compared to plant material. This enrichment is likely attributed to the accumulation of 15N during the decomposition of the deposited material. This study confirms the possibility of tracking changes in the aquatic vegetation of softwater lakes based primarily on the sediment δ13CORG values along the alkalization gradient.