The extraction of metal from parent ore is influenced by many factors including microbial ones. It is now clear that microorganisms provide the space for biological and chemical reactions to occur and produce the necessary ferric ions and protons for leaching. Microbial dynamics within bio-hydrometallurgical and bio-oxidation processes are critical to their performances, and a fundamental understanding of key microbial species within such processes is important throughout the lifetime of their operations. Quantitative analysis of the active microbial communities during the cultivation of a mesophilic culture is reported in this study. Cell concentration and microbial community changes were measured and analyzed over 6 weeks of culture maintenance with a fresh medium. It was observed that a culture previously dominated by Leptospirillum ferriphilum was dominated by Ferroplasma acidiphilum and Acidiplasma cupricumulans species after 2 weeks, while Thermoplasmatales sp. and Cuniculiplasma divulgatum dominated the culture for the remainder of the 4-week of operation. The observed decrease in biomass concentration corresponded to the resultant decrease in ferrous-ion oxidation during the maintenance period. This shift in the microbial community structure was thought to be catalyzed by the constituent of the medium, which is indicative of the microbial adaptability of the individual strains in the culture to changes in the medium composition. Thus, this study may provide insight into the activity of these microbes to better harness their functionality by changing process conditions. This understanding may help in overcoming potential technical challenges associated with the processing of low-grade and complex ores due to microbial dynamics.