Bioleaching as a method for extracting metals from low-grade ores is gaining attention due to its potential as an alternative to energy-intensive and less-efficient metallurgical processes. Furthermore, increasing the chloride concentration in bioleaching environments may enhance the efficiency, thus raising the interest also in halotolerant acidophilic iron oxidizers like Sulfobacillus thermosulfidooxidans. This study aimed to compare the chloride tolerance of various Sulfobacillus species. The results showed that Sb. thermosulfidooxidans exhibited the highest tolerance compared to other tested strains. Based on the knowledge, that yeast extract (YE) may have a positive impact on the chloride tolerance of microorganisms, the importance of single YE components on bacterial respiratory activity was also investigated. The experiments showed that in Sb. thermosulfidooxidans casamino acids with potassium and glucose can be applied to replace the yeast extract. Moreover, ultrafiltration of casamino acids indicated the importance of small peptides or amino acids (<1 kDa). This study contributes to the understanding of the requirements for creating a defined medium for an efficient bioleaching process in the presence of chloride ions involving Sb. thermosulfidooxidans.