Tufa is a fresh-water surface calcium carbonate deposit precipitated at or near ambient temperature, and commonly contains the remains of macro- and microphytes. Many Holocene tufas are found along the Zrmanja River, Dalmatian karst, Croatia. In this work we present radiocarbon dating results of older tufa that was found for the first time at the Zrmanja River near the Village of Sanaderi. Tufa outcrops were observed at different levels, between the river bed and up to 26 m above its present level. Radiocarbon dating of the carbonate fraction revealed ages from modern, at the river bed, up to 40 kBP ~20 m above its present level. These ages fit well with the hypothesis that the Zrmanja River had a previous surface connection with the Krka River, and changed its flow direction toward the Novigrad Sea approximately 40 kBP (Marine Isotope Stage 3). Radiocarbon AMS dating of tufa organic residue yielded a maximum conventional age of 17 kBP for the highest outcrop position indicating probable penetration of younger organic material to hollow tufa structures, as confirmed by radiocarbon analyses of humin extracted from the samples. Stable carbon isotope composition (δ13C) of the carbonate fraction of (−10.4 ± 0.6)‰ and (−9.7 ± 0.8)‰ for the Holocene and the older samples, respectively, indicate the autochthonous origin of the carbonate. The δ13C values of (−30.5 ± 0.3)‰ and (−29.6 ± 0.6)‰ for organic residue, having ages <500 BP and >5000 BP, respectively, suggest a unique carbon source for photosynthesis, mainly atmospheric CO2, with an indication of the Suess effect in δ13C during last centuries. The oxygen isotopic composition (δ18O) agrees well with deposition of tufa samples in two stages, the Holocene (−8.02 ± 0.72‰) and “old” (mainly MIS 3 and the beginning of MIS 2) (−6.89 ± 0.34‰), suggesting a ~4 °C lower temperature in MIS 3 compared to the current one.