Submerged macrophytes play a key role in freshwater ecosystems by influencing carbon and nitrogen cycling. Stable isotope analyses (δ¹³C, δ¹⁵N) of macrophyte bulk organic matter provide valuable insights into the biogeochemical processes shaping these cycles. We investigated the isotopic signatures of vascular macrophytes (elodeids) and macroscopic green algae (charophytes) across lakes differing in depth and mixing regimes. A general trend of ¹²C enrichment in macrophyte organic matter with increasing depth was observed, consistent for both elodeids and charophytes. This pattern was less pronounced in lakes with limited depth gradients, where mixing likely homogenized the availability of inorganic carbon sources. Nitrogen isotopic signals were more variable: Elodea canadensis in Lake Czyste and Stuckenia pectinata in Lake Borzyszkowskie showed increasing δ¹⁵N with depth, whereas Nitella flexilis in Lake Czyste exhibited the opposite trend. In Lake Dymno, S. pectinata displayed no clear depth-related pattern for δ¹⁵N, underscoring species-specific and lake-specific responses. To identify the environmental drivers of isotopic variability, we applied multivariate analyses. Principal Component Analysis indicated that depth played a crucial role for δ¹³C, while δ¹⁵N was most strongly correlated with Ca²⁺, pH, and conductivity, parameters typically associated with hardwater lakes. Variation partitioning analysis further showed that water chemistry variables (pH, Ca²⁺ concentration) explained 30% of the total variation, environmental variables (depth, photosynthetically active radiation) explained 9%, whereas nutrient-related parameters (total nitrogen and total phosphorus) explained only 3% of measurable variation. Our findings demonstrate that physical processes predominantly influence δ¹³C, whereas δ¹⁵N variability reflects chemical conditions related to lake hardness and redox processes, thus enhancing nitrification or denitrification processes. These results highlight the complexity of isotopic responses in submerged macrophytes and the need for further research into the environmental mechanisms controlling isotopic signatures in freshwater ecosystems.