This study aimed to investigate the hexachlorobenzene (HCB) dechlorinating ability of sediment microbes collected from a natural canal receiving secondary effluents from an industrial estate and nearby factories. Nine sites along the stream and one in the estuary in the Gulf of Thailand into which the canal spills were specified and sampling for sediment and water. Preliminary analysis of the sediments showed that the first four sites nearest to the discharging location were contaminated by HCB within the range of 0.18 to 1.25 ppm. Apart from that, 1,3,5-trichlorobenzene which has never been commercially produced or used in any manufacturing processes except for the transformation from higher chlorinated benzene was also identified in the range of 0.16 to 0.24 ppm. This suggested a possibility of sporadically HCB contamination in this stream. Of more important, people in the community along this canal earn their living by coastal fishery; hence, posing a risk of spreading HCB and its less chlorinated congeners via food chain from caught marine creatures to human. As a result, there is an urgent need to understand the behavior of HCB dechlorination in this stream sediment which can lead to a clean-up action in the future. Serum bottles with sediment slurries (sediment to water ratio of 1:1 (v/v) and filtered to remove particles larger than 0.7 mm) from each site were inoculated with 2 mg/l of HCB, kept anaerobically in the dark at room temperature without any nourishment, and analyzed for HCB and its less-chlorinated congeners every 6 days. Total chemical oxygen demand, suspended solids, and volatile suspended solids were in the range of 21,492-73,584, 158,100-518,100 and 6,000-32,700 mg/l, respectively. It was found that all sediment slurries began to dechlorinate HCB in 12 to 30 days and the HCB was completely removed within 42 to 60 days or so. On the other hand, there was no HCB dechlorination occurred in the controlled set which was sterilized by autoclaving prior to the addition of HCB. This implies that the HCB transformation was solely due to microorganisms’activities. HCB was dechlorinated principally via pentachlolobenzene to 1,2,3,5-tetrachlorobenzene and terminated at 1,3,5-trichlorobenzene which is the major pathway as reported by many researchers. Dichlorobenzene has not been detected in any samples within the dechlorination period of 60 days. The results indicate that the microbial matrix in the sediment of this stream has an outstanding capability to dechlorinate HCB. Existing substrates and nutrients which mainly sorbed onto the solid phase and the typical temperature in Thailand were sufficient and suitable to promote the activities of these HCB-dechlorinating microbes.