Thrombolite and stromatolite, as typical representatives of microbialites, contain biotic minerals that significantly influence their unique characteristics. However, a key problem for distinguishing between biotic and abiotic minerals by thermodynamic characteristics has confused researchers for some time. For this study, thirty-two samples including wormkalk, micrite, thrombolite, and stromatolite were collected from Sinian in Suzhou, Anhui Province, China. Mineral morphologies and elemental compositions were analyzed by Scanning electron microscopy (SEM)-Energy dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FT-IR). The results of SEM showed that the appearance of spherical and acicular shapes may be indicative of biotic minerals in thrombolite and stromatolite. The results of FT-IR showed that the phosphorus and organic functional groups were also discovered. Mineral constituent and crystal structure were analyzed by X-ray diffraction, which revealed that calcite was the dominant mineral. However, the crystallinity degree of thrombolite and stromatolite was higher compared to wormkalk and micrite. The stable carbon isotope ratios revealed that the delta 13C values of thrombolite and stromatolite ranged from -6.71 parts per thousand to -4.32 parts per thousand, which were more negative than those of wormkalk and micrite (1.03 parts per thousand to 2.15 parts per thousand). Comparative studies of thermodynamic characteristics were carefully investigated using thermogravimetric analysis. Results showed that the thermostability and pyrolysis temperature of thrombolite and stromatolite were clearly higher. Therefore, important insights were provided to understand the unique characteristics of biotic minerals in thrombolite and stromatolite. A reliable and quantitative reference index was established to further distinguish between biotic and abiotic minerals.