Sulfate-reducing bacteria are known to mediate dolomite formation under hypersaline conditions, but details of the crystal nucleation process are still poorly constrained. Our laboratory study demonstrates for the first time that Desulfobulbus mediterraneus, a marine sulfate-reducing bacterium, mediates primary precipitation of Mg-rich dolomite under anoxic conditions in media replicating modern seawater chemistry at low temperature (21 °C). Precipitation of crystals was associated with extracellular polymeric substances in a monospecific biofilm, providing templates for nucleation by altering the molar Mg/Ca ratio. After initial nucleation of single nanospherulites (∼50 nm), growth was mediated by aggregation, resulting in spherulites of ∼2–3 μm in diameter. Nucleation led to differences in Mg/Ca ratios and δ44/40Ca values among the organic material (i.e., biofilm including cells and extracellular polymeric substances; 0.87 ± 0.01 [2 SD] and 0.48‰ ± 0.11‰ [2 SE], respectively), the crystals (1.02 ± 0.11 [2 SD] and <−0.08‰ ± 0.24‰ [2 SE], respectively), and the liquid bulk medium after mineral precipitation (4.53 ± 0.04 [2 SD] and 1.10‰ ± 0.24‰ [2 SE], respectively). These data indicate a two-step fractionation process involved in the sequestration of Ca from the solution into the crystal lattice of the mineral precipitated. Our results demonstrate the capability of extracellular polymeric substances to overcome kinetic inhibition, fostering the formation of kinetically less favorable Mg-rich dolomite, and they also question the applicability of the Ca isotopic system as a proxy for paleogeochemistry of seawater.