Subsurface granitic environments are scarce and poorly investigated. A multidisciplinary approach was used to characterize the abundant moonmilk deposits and associated microbial communities coating the granite walls of the 16th Century Paranhos spring water tunnel in Porto city (north-west Portugal). It is possible that this study is the first record of moonmilk in an urban subsurface granitic environment. The morphology and texture, mineralogical composition, stable isotope composition and microbial diversity of moonmilk deposits have been studied to infer the processes of moonmilk formation. These whitish secondary mineral deposits are composed of very fine needlefibre calcite crystals with different morphologies and density. Calcified filaments of fungal hyphae or bacteria were distinguished by field emission scanning electron microscopy. Stable isotope analysis revealed a meteoric origin of the needle-fibre calcite, with an important contribution of atmospheric CO 2 , soil respiration and HCO À 3 from weathering of Ca-bearing minerals. The DNAbased analyses revealed the presence of micro-organisms related to urban contamination, including Actinobacteria, mainly represented by Pseudonocardia hispaniensis, Thaumarchaeota and Ascomycota, dominated by Cladosporium. This microbial composition is consistent with groundwater pollution and contamination sources of the overlying urban area, including garages, petrol stations and wastewater pipeline leakage, showing that the Paranhos tunnel is greatly perturbed by anthropogenic activities. Whether the identified microorganisms are involved in the formation of the needle-fibre calcite or not is difficult to demonstrate, but this study evidenced both abiotic and biogenic genesis for the calcite moonmilk in this subsurface granitic environment.