The Rio Naracauli in SW Sardinia drains part of the Ingurtosu Zn–Pb mining district, and contains extreme concentrations of dissolved Zn at near-neutral pH. In the upper reaches of the stream, pH, alkalinity and Zn concentrations are such that hydrozincite [Zn₅(CO₃)₂(OH)₆] precipitates in a biologically mediated process facilitated by a microalga (Chlorella sp.) and a cyanobacterium (Scytonema sp.). Values of δ⁶⁶Zn in water and solid samples ranged from − 0.35‰ to + 0.5‰ relative to the JMC 3-0749-Lyon standard, and closely follow a mass-dependent fractionation line. Two composite samples of sphalerite, the primary ore mineral in the Ingurtosu deposits, had an average δ⁶⁶Zn of + 0.15‰, similar to sphalerite measured elsewhere in hydrothermal mineral deposits. Zinc isotope measurements of the stream water and the hydrozincite forming in the stream show a consistent preference for the heavy isotope, ⁶⁶Zn, in the hydrozincite relative to ⁶⁴Zn. Synthetic hydrozincites produced without added bacteria have δ⁶⁶Zn identical to the dissolved Zn, thus suggesting a biologically mediated mineralization process in Rio Naracauli. The average fractionation, Δ_hdz-water, is 0.35‰, the magnitude of which is consistent with other studies, and suggests an extracellular mechanism of the biomineralization process. Zinc concentration and dissolved δ⁶⁶Zn steadily decrease in the reach of the stream where the biomineralization occurs. The biomineralization process also leads to the sequestration of Pb, Cu and Ni in the hydrozincite lattice, and the coeval precipitation of an amorphous CdCO₃ solid, prompting the suggestion that if optimized, the biomineralization process might represent a feasible passive remediation strategy for streams with high Zn and other metals, and with near-neutral pH.