The effect of physicochemical conditions (residence time, oxygen concentrations, and chalk characteristics) on the biodegradation of 2,4,6-tribromophenol (TBP) during transport was investigated in low-permeability fractured-chalk cores. Long-term (approximately 600 d) biodegradation experiments were conducted in two cores (approximately 21 cm diameter, 31 and 44 cm long, respectively), intersected by a natural fracture. TBP was used as a model contaminant and as the sole carbon source for aerobic microbial activity. Bacterial isolates were recovered and identified by both Biolog identification kit and 16S rDNA sequences from batch enrichment cultures. One of the strains, with 98% similarity (based on the 16S rDNA data) to Achromobacter xylosoxidans, was shown to have the ability to degrade TBP in the presence of chalk. The decrease in TBP concentration along the fracture due to biodegradation was not affected by reducing the residence time from 49 to 8 min. In contrast, adding oxygen to the water at the inlet and increasing the flow rates improved TBP removal. Although the matrix pore-size distribution limits microbial activity to the fracture void, the chalk appears to provide an excellent environment for biodegradation activity. Approximately 90% of TBP removal occurred within 10 cm of the TBP source, indicating that in-situ bioremediation can be used to remove organic contaminants in low-permeability fractured rocks if nutrient-delivery pathways within the aquifer are secured.