Microbial anaerobic dechlorination of hexachlorobenzene (HCB) was examined in sediment slurries amended with two classes of nonionic surfactant, polyoxyethylene (POE) sorbitan fatty acid esters (Tweens) and POE alcohols (Brijs). The rationale for surfactant addition was to increase the bioavailability of highly sorbed organic pollutants to degrading microorganisms by enhancing their solubility. The solubility of HCB was initially enhanced via micellar partitioning; however, primary degradation of most surfactants occurred within 10 d. Dechlorination activity was significantly reduced at POE alcohol concentrations above the critical micelle concentration (cmc), with or without the occurrence of surfactant degradation. Tween 80 decreased HCB dechlorination at concentrations significantly above the cmc (1,500–5,000 mg/L). At concentrations closer to the cmc (300–1,200 mg/L), Tween 80 increased dechlorination rate constants four‐ to fivefold in acclimated slurries. Additions of Tween 80 at or below the cmc (3, 30, 300, and 911 mg/L) stimulated dechlorination activity in unacclimated slurries that exhibited very little activity in unamended controls. An average of 89% of HCB was dechlorinated after 90 d, compared to 20% in unamended sediments. No effect was observed for POE alcohols at these sub‐cmc levels. The lack of a stimulated response for the POE alcohols suggests that Tween 80 may not be acting simply as a source of carbon or energy.