The tufas of the presently shallow Chlupáčova sluj Cave document an unusual history of deeper to shallower hydrologic processes in cave evolution. The wider geological context of the cave, along with fluid inclusion and stable isotope (C and O) analyses of calcite from tectonic veins cutting through its wallrock, are evidence of the origin of the cave in the deeper subsurface, under the influence of ascending 25–70 °C warm, saline (2.0–22.3 wt% eq. NaCl) waters driven by regional hypogene processes. The formation of calcite veins, which occurred at 700–400 ka, was probably coeval with the early stages of cave development. However, two generations of tufa formed in the later Pleistocene during the mature shallow-subsurface stage of cave development when daylight and cold meteoric waters penetrated the cave through ceiling windows and tectonic fractures.The first-generation tufa, which consists of cauliflower-like aggregates of hollow, outward-radiating carbonate tubules covered with isopachous banded calcite cements, is interpreted as ancient bryophyte tufa that was deposited about 620 ± 140–530 ± 110 ka. The presence of microscopic biomorphic fabrics resembling micro-stromatolites, calcareous algal chambers, calcite crystals with cloudy cores and clear sparitic rims, and specific biomarkers identified in tufa extracts suggest that, in addition to mosses, algae and bacteria may have also been instrumental in promoting the deposition of tufa. Tufa stable isotope (C and O) characteristics and fluid inclusion contents point to deposition of this first-generation tufa from fresh water with a minor admixture of higher hydrocarbons at the surface temperature.Post-dating the first-generation tufa, minute grains of exotic minerals (e.g. stibnite, gothic-arch calcite, gypsum, Mn-minerals, opaline silica) and sparry calcite were precipitated in the tufa moulds and between carbonate tubules as cements. These minerals suggest at least one event in which hydrothermal waters entered the cave from below after deposition of the first tufa from cool meteoric waters. In this hydrothermal phase, the cave may have been a hot spring.The second-generation tufa originated during the Riss – Würm Interglacial at 121 ± 2.1–107 ± 2.6 ka, following a period of intense water erosion that partly erased older spelean deposits from cave walls. This tufa consists of highly porous, friable, crudely laminated carbonate sediments with floral and molluscan remains, characteristic of common cold-water karstic tufa. This and the earlier tufa were, in turn, covered by later fine-grained cave sediments, contributing to the preservation of Pleistocene tufa that records an alternation of deep and shallow waters in the cave over at least the last 500,000 years.