The geochemistry and isotopic composition (H, O, S, O_sulfate, C, Sr) of groundwater from the Nubian Sandstone (Kurnub Group) aquifer in the Negev, Israel, were investigated in an attempt to reconstruct the origin of the water and solutes, evaluate modes of water–rock interactions, and determine mean residence times of the water. The results indicate multiple recharge events into the Nubian sandstone aquifer characterized by distinctive isotope signatures and deuterium excess values. In the northeastern Negev, groundwater was identified with deuterium excess values of ∼16‰, which suggests local recharge via unconfined areas of the aquifer in the Negev anticline systems. The δ¹⁸O_H2O and δ²H values (−6.5‰ and −35.4‰) of this groundwater are higher than those of groundwater in the Sinai Peninsula and southern Arava valley (−7.5‰ and −48.3‰) that likewise have lower deuterium excess values of ∼10‰. Based on the geochemical differences between groundwater in the unconfined and confined zones of the aquifer, a conceptual geochemical model for the evolution of the groundwater in the Nubian sandstone aquifer has been reconstructed. The isotopic composition of shallow groundwater from the unconfined zone indicates that during recharge oxidation of pyrite to SO₄ (δ³⁴S_SO4 ∼−13‰; δ¹⁸O_SO4 ∼+7.7‰) and dissolution of CaCO₃ (⁸⁷Sr/⁸⁶Sr ∼0.70787; δ¹³C_DIC = −3.7‰) occur. In the confined zone of the aquifer, bacterial SO₄ reduction removes a significant part of dissolved SO42-, thereby modifying its isotopic composition (δ³⁴S_SO4 ∼−2‰; δ¹⁸O_SO4 ∼+8.5‰) and liberating dissolved inorganic C that contains little or no radiocarbon (¹⁴C-free) with low δ¹³C_DIC values (<−12‰). In addition to local recharge, the Sr and S isotopic data revealed contribution of external groundwater sources to the Nubian Sandstone aquifer, resulting in further modifications of the groundwater chemical and isotopic signatures. In the northeastern Negev, it is shown that SO₄-rich groundwater from the underlying Jurassic aquifer contributes significantly to the salt budget of the Nubian Sandstone aquifer. The unique chemical and isotopic composition of the Jurassic groundwater (δ³⁴S_SO4 ∼ +14‰; δ¹⁸O_SO4 ∼ 14‰; ⁸⁷Sr/⁸⁶Sr ∼0.70764) is interpreted as reflecting dissolution of Late Triassic marine gypsum deposits. In the southern Arava Valley the authors postulate that SO₄-rich groundwater with distinctively high Br/Cl (3 × 10⁻³) low ⁸⁷Sr/⁸⁶Sr (0.70734), and high δ³⁴S_SO4 values (+15‰) is derived from mixing with underlying brines from the Paleozoic units. The radiocarbon measurements reveal low ¹⁴C activities (0.2–5.8 pmc) in both the northeastern Negev and southern Arava Valley. Taking into account dissolution of carbonate rocks and bacterial SO₄ reduction in the unconfined area, estimated mean residence times of groundwater in the confined zone in the northeastern Negev are on the order of 21–38 ka, which suggests recharge predominantly during the last glacial period. The ¹⁴C signal in groundwater from the southern Arava Valley is equally low but due to evidence for mixing with external water sources the residence time estimates are questionable.