The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO3) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c. 80% of the EPS were turned over in the upper 15–20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5–8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO3. We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO3 to precipitate. Therefore, this model explains interactions between EPS and CaCO3 precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.