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Objective: The work aimed to isolate and culture the acidophilic and moderately thermophilic microorganisms for leaching the sulfide ore., Methods: We enriched and incubated iron- or sulfur-oxidizing strains from muddy water of acuric hot spring utilizing ferrous irons or elemental sulfur as substrates. Then, we identified the strains by their morphological, physiological, biochemical properties and phylogenetic positions, and estimated their bioleaching potential according to their oxidation rate of pyrite., Results: Two acidophilic, aerobic and facultative heterotrophic bacterial strains, Costa C and Costa E, were isolated from the samples of sulfuric hot springs of Costa Rica. Cells of the two strains were gram-positive, spore-forming and rod-shaped [(0.4 - 0.6) microm x (2.5 - 4.0) microm and (0.4 - 0.7) microm x (2.4 - 4.9) microm, respectively]. Strain Costa C grew at a temperature range of 30 degrees C - 55 degrees C and a pH range of 1.2 - 5.0, optimally at 50 degrees C and 2.8. Strain Costa E grew at a temperature range of 30 degrees C - 55 degrees C and at a pH range of 1.4 - 5.0, optimally at 40 degrees C and 2.8. Two strains could autotrophically grow on inorganic substrates such as ferrous irons, element sulfur and K2 S4 O6 and also could utilize organic substrates like yeast extract for heterotrophic growth. Phylogenetic analysis based on 16S rRNA gene sequences alignment demonstrated that the highest similarity between strain Costa C, Costa E and other species of the genus Sulfobacillus was above 99%., Conclusion: Based on morphological, physiological and biochemical analysis, Costa C and Costa E can be affiliated to the genus Sulfobacillus, for which the names Sulfobacillus sp. strain Costa C and Sulfobacillus sp. strain Costa E were proposed. Both strains could oxidize pyrite, and the oxidation rates arrived 63.0 mg/L x d and 56.8 mg/L x d, respectively.