Biological clogging is promoted in aquifers either to contain or to remediate groundwater. In this study, an apparatus able to detect small changes in hydraulic conductivity (K) was developed to measure the clogging of a single fracture in limestone, following microbial stimulation. The fracture had a 2.5 mm2 section and was 50 cm long. Prior to the inoculation of the limestone, the sequencing of representative clones from 16S rRNA genes isolated from groundwater, showed significant affiliation with Cytophaga spp., Arcobacter spp. and Rhizobium spp. These bacteria are known to secrete extracellular polymeric substances and form biofilms. When nutrients were added to the inoculated limestone, a decrease in K occurred after 8 days, reaching 0.8% of its initial value after 22 days (Kfi = 340 cm min-1). This study showed that a stimulation of indigenous microbes from groundwater effectively clogged a macrofracture in limestone, suggesting the potential application of biobarriers in fractured rock aquifers.