Molecular and microbiological analysis of a laboratory bioreactor biomass oxidizing thiocyanate at autotrophic conditions and at 1 M NaCl showed a domination of a single chemolithoautotrophic sulfur-oxidizing bacterium (SOB) capable of using thiocyanate as an energy source. The bacterium was isolated in pure cultures and identified as a member of the Halothiobacillus halophilus/hydrothermalis clade. This clade includes moderately halophilic chemolithoautotrophic SOB from marine and hypersaline habitats for which the ability to utilize thiocyanate as an electron donor has not been previously demonstrated. Halothiobacillus sp. strain SCN-R1 grew with thiocyanate as the sole energy and nitrogen source oxidizing it to sulfate and ammonium via the cyanate pathway. The pH range for thiocyanate oxidation was within a neutral region between 7 and 8 and the range of salinity was from 0.2 to 1.5 M NaCl, with an optimum at 0.5 M. Despite the close phylogenetic relatedness, none of the tested type strains and other isolates from the H. halophilus/hydrothermalis group exhibited thiocyanate-oxidizing capacity.