The application of tritium, 2H, and 18O in the characterization of the precipitation, groundwater, and surface and lake water of the Plitvice Lakes (PL), Croatia, over the 1979–2019 period is presented. An increase in the mean annual air temperature of 0.06 °C/year and in the annual precipitation amount of 10 mm/year is observed. The good correlation of the tritium activity concentration in the PL and Zagreb precipitation implies that the tritium data for Zagreb are applicable for the study of the PL area. The best local meteoric water line at PL was obtained by the reduced major axis regression (RMA) and precipitation-weighted ordinary least squares regression (PWLSR) approaches: δ2HPWLSR = (7.97 ± 0.12) δ18O + (13.8 ± 1.3). The higher deuterium excess at PL (14.0 ± 2.2 ‰) than that at Zagreb reflects the higher altitude and influence of the Mediterranean precipitation. The δ2H in precipitation ranges from −132.4‰ to −22.3‰ and δ18O from −18.3 ‰ to −4.1‰. The much narrower ranges in the groundwater (<1‰ in δ18O, <10‰ in δ2H) indicate the good mixing of waters in aquifers and short mean residence times. The higher average δ2H in all three karst springs observed after 2003 can be attributed to the increase in the mean air temperature. The mean δ2H and δ18O values in the surface and lake water increase downstream due to the evaporation of surface waters. There is no significant difference between the surface water line and the lake water line (2011–2014). The stable isotope composition of the surface and lake waters reacts to extreme hydrological conditions.