Tufas are freshwater carbonate rocks that form in continental environments through a combination of physical, chemical, and biological processes. This study investigates the role of microorganisms in the precipitation of Quaternary tufa deposits in the Serra da Bodoquena Formation, in the Bonito region. Two sites along the Mimoso River, named Taíka and Mimosa, characterized by the pool–barrage–cascade depositional subenvironment, were selected for this study. Four distinct facies were identified: stromatolitic boundstones, phytoherm boundstones of algae, phytoherm boundstones of bryophytes, and phytoclastic rudstones. These facies were observed in diverse hydrological settings, including fast-flowing waters, such as waterfalls and cascades, as well as slow-flowing areas, such as pools and dams. The δ18O depletion indicated a meteoric origin for the fluid involved in carbonate precipitation. The low δ13C values were attributed to photosynthetic processes and the contribution of light carbon-enriched groundwater. The presence of Oocardium stratum and calcified organic mucilage from extracellular polymeric substance (EPS) corroborates the significant role of microorganisms in tufa formation, particularly in stromatolitic boundstones and phytoherm boundstones of algae. Rapid CO2 degassing significantly contributes to mineralization in fast-flowing waters. Micro-CT results offer detailed insights into the relationship between mechanical processes and biological influences in shaping porosity characteristics. The findings of this study significantly enhance our understanding of the role of microorganisms in tufa formation, highlighting the complex interplay between biotic and abiotic processes in the development of different tufa facies. Moreover, the insights gained from this study provide valuable implications for interpreting tufa deposits worldwide.