The Zagreb aquifer is the main source of potable water for the inhabitants of the City of Zagreb and Zagreb County. It presents a strategic water reserve protected by the Republic of Croatia. All previous studies related to the definition of the groundwater–surface interaction in the study area have been made based on the isotopic composition of the Sava River from the location of the Domovinski Most bridge, which is located downstream of most pumping well fields. In 2019, a new monitoring station was established at the Podsusedski Most bridge, at the entrance of the Sava River into the Zagreb aquifer, approximately 23 km upstream of the Domovinski Most bridge. Within this research, water isotope data (δ2H, δ18O, deuterium excess) from both Sava River and groundwater sites were used along with hydrologic data to examine the extent to which hydrologic conditions affect the isotope signature and whether the interaction between groundwater and the Sava River causes a change in the isotopic composition of the Sava River. In addition, δ18O amplitudes were estimated for different time periods, as well as the mean residence time for the hydrological year 2019/2020. For that purpose, different statistical methods were applied to the new monthly data for six years for the Domovinski Most bridge and two years for the Podsusedski Most bridge. The δ18O amplitudes vary from 0.22 to 1.86 depending on the time interval and hydrological conditions, while the mean residence time for the hydrological year 2019/2020 was estimated to be about 2.5 months.