We investigated changes in the chemical characteristics of evaporating seawater under the influence of microbial activity by conducting geochemical analyses of the brines and evaporite sediments collected from solar salterns in Trapani, Italy. The microbial activity had a substantial effect on the carbonate system parameters. Dissolved inorganic carbon (DIC) was substantially removed from the brine during the course of evaporation from the seawater to the point where calcium carbonate precipitates, with an accompanying decrease in its carbon isotopic composition (δ13CDIC) to as low as −10.6‰. Although the removal of DIC was due to calcium carbonate precipitation, photosynthesis, and the degassing of CO2(aq) induced by evaporation, the presence of 13C-depleted δ13CDIC in ponds where calcium carbonate precipitates can be attributed to the dissolution of atmospheric CO2 because of intensive CO2(aq) uptake by photosynthesis, and/or mineralization of organic matter by sulfate reduction. In contrast, δ13CDIC increased up to 7.2‰ in the salinity range where halite precipitates, which can be ascribed to the domination of the effect of degassing of CO2(aq) under conditions with reduced microbial activity. A gradual decrease in microbial activity was also reflected in compound-specific δ13C of photosynthetic pigments; isotopic fractionation associated with DIC assimilation increased linearly as the evaporation proceeded, indicating DIC-limited conditions within the microbial mats and gypsum crusts because of restricted DIC diffusion from the overlying brine and/or suppression of primary production at higher salinity.