In this paper, we analyze the background activity of anthropogenic radionuclides (14C, 3H, 137Cs, and 239,240Pu), emphasizing 14C content, in terrestrial and aquatic ecosystems in the Lithuanian border region before the commissioning of a new nuclear power plant in Belarus (BelNPP). In terrestrial samples, the 14C concentration varied insignificantly—from 98.6 ± 0.7 to 102.2 ± 0.8 pMC, which is close to the 14C level in atmospheric CO2. In aquatic samples, the 14C concentration varied within wide limits from 76.9 ± 0.7 to 99.6 ± 0.6 pMC, depending on the ecological group of macrophytes. Various ecological groups of macrophytes have experienced the influence of a freshwater reservoir effect. This lowest 14C content in submerged macrophyte species, within the limits of uncertainty, was very close to the specific activity of 14C in DIC (78.6 ± 0.6 pMC) in the water of the Neris River. The background 14C values, together with the data on 3H, 137Cs and 239,240Pu obtained in this study, can be used in the future to assess the contribution of the BelNPP conventional radioactive effluents to the levels of 14C and other radionuclides in terrestrial and aquatic ecosystems of the transboundary region of Belarus and Lithuania.