In a phytotron experiment, the effects of pH variation and eutrophication on isoetids plants from soft-water lakes specifically the submerged form of Littorella uniflora (L.) Asch. was investigated by analyzing stable carbon and nitrogen isotopes (δ13C and δ15N). Conducted in late October 2020, 200 specimens from Lake Zawiad, near Gdansk, Poland, were examined over 75 days. The study tested three pH levels (∼4.5, ∼7.0, and ∼8.5) and a detailed 12-step nutrient gradient (nitrogen: 0–10 mg/l; phosphorus: 0–0.3 mg/l). The analysis focused on isotopic composition in leaves and roots, revealing that acidic conditions favored higher δ13C values (leaves: −22.67 ‰; roots: −23.23 ‰), suggesting a preference for lighter carbon forms in photosynthesis and intensive use of limited sources of CO2. The neutral pH variant showed the lowest δ13C values (leaves: −25.53 ‰; roots: −25.47 ‰), indicating less optimal conditions. δ15N values exhibited minimal fluctuation across pH levels, with slight variations in acidic and alkaline environments compared to neutral conditions. An observed decrease in δ13C across all pH levels with increased nutrients, alongside a rise in δ15N values, indicates a complex interaction between isotopic composition and environmental factors. Our findings suggest that L. uniflora shows a distinct isotopic response to varying pH levels, with higher δ13C values under acidic conditions potentially indicating enhanced CO2 uptake through a specialized carbon assimilation strategy. This highlights the species' adaptive mechanisms to environmental stressors, suggesting that the isotopic composition of aquatic vegetation can serve as a sensitive indicator of changes in lake ecosystems.