Salinity negatively affects growth of sulfur-oxidizing bacteria (SOB) and their sulfate production ability, meanwhile decreases the available sulfate for plants in soil. The aim of this study was to isolate and characterize the bacteria of genus Halothiobacillus, as a salt-tolerant SOB, from saline and sulfidic habitats of Iran for the first time and evaluating the effect of salinity on their biomass and sulfate production during the oxidation of different sulfur sources. Isolation process and surveying the morphological, biochemical and 16S rRNA gene analysis resulted into identification of three species (eight strains) of Halothiobacillus genus including H. neapolitanus, H. hydrothermalis and H. halophilus. Salinity (0, 0.5, 1, 2 and 4 M NaCl) had a significant impact (p ≤ 0.01) on bacterial biomass and sulfate production during the oxidation of thiosulfate and elemental sulfur. Biomass and sulfate production by strains was accompanied by a decrease in residual content of thiosulfate (RCT) in medium. The amount of produced biomass and sulfate in medium supplemented by thiosulfate was much higher than elemental sulfur. The highest amount of biomass and sulfate was produced by H. neapolitanus strain I19 at 0.5 and 1 M NaCl concentration. The results of this study could be the first step to focus on the application of these bacteria to increase sulfate storage of saline soils and crop production.