The mechanism of sedimentary dolomite formation has puzzled the geology community for more than a century. Within the past several years, successful synthesis of disordered dolomite under ambient conditions using abiotic materials derived from microbial organisms such as polysaccharides and exopolymeric substances (EPS) has been reported. The success in laboratory experiments has driven this study to find evidence in natural ancient carbonate samples that correlate dolomite formation and the presence of organic matter. A micro-laminated carbonate with alternating dolomite–calcite layers from the mid-Lower Ordovician St. Paul Group from the Central Appalachians in southern Pennsylvania was examined using optical microscopes, X-ray diffraction (XRD), scanning electron microscopy (SEM) with X-ray energy-dispersive spectroscopy, electron microprobe analysis (EPMA), scanning transmission electron microscopy (STEM), laser-induced fluorescence (LIF) imaging, short-wave infrared (SWIR) imaging, and X-ray fluorescence (XRF) imaging. The sample is composed mainly of two types of layers. Dolomite-dominated layers are darker in color, generally thinner, and contain detrital minerals such as quartz and feldspar. In contrast, calcite-dominated layers are lighter in color, thicker, and contain less detrital minerals supported by microcrystalline calcite matrix. In situ XRD, LIF, XRF, and SWIR results show that organic remnants are enriched in the dolomite layers. The coincided spatial distribution confirmed a positive correlation between dolomite and organic matter, and hence provide evidence for microbial-EPS-catalyzed formation of sedimentary dolomite.