Microorganisms with importance to mineral biotechnology are commonly maintained through live culture maintenance, which can be labour intensive and expensive. Live culture maintenance can also result in contamination, genetic drift and loss of traits, or whole strains, that are crucial to some biotechnological applications. This study aimed to investigate alternative preservation methods, and to determine the best method required for the preservation of mixed microbial cultures capable of bioleaching chalcopyrite under salt stress. Live culture maintenance followed by cold storage at 4 °C, liquid drying and temperature-controlled cryopreservation were used to preserve mesophilic (30 °C), moderately thermophilic (45 °C) and thermophilic (60 °C) bioleaching cultures. Recovery of cells and bioleaching activity were determined following revival. Results were compared to cultures routinely maintained by subculturing. Across all temperatures, cryopreservation with 10% (v/v) glycerol as a cryoprotectant resulted in the highest post-revival cell recovery. Further research is required to determine if the microbial diversity in each culture is impacted by the preservation method employed.