The Ediacaran Doushantuo Formation (DST) is renowned for exceptionally preserved Precambrian fossils including metazoans. Some of these fossils, particularly microfossils such as multicellular algae and acanthomorphic acritarchs, are preserved in DST chert nodules. To better understand the geomicrobiological processes that contributed to the authigenic formation of DST chert nodules and facilitated exceptional fossil preservation, we analyzed organic matter in these chert nodules and the surrounding matrix (calcareous mudstone) using multiple in-situ techniques: confocal laser Raman spectroscopy, micro-Fourier transform infrared spectroscopy (FTIR), and secondary ion mass spectroscopy (SIMS). We found strong ultrastructural, chemical, and isotopic heterogeneities in the organic matter as indicated by the Raman spectral parameter I-1350/1600 ranging from 0.49 to 0.88, the infrared spectral index R3/2 from 0.12 to 0.90, and an estimated δ13Corg-SIMS range of 44‰ (V-PDB). These micron-scale heterogeneities imply that the organic matter preserved in the DST chert nodules is derived from different carbonaceous sources in a diverse microbial ecosystem, including eukaryotic and/or prokaryotic photoautotrophs, as well as chemotrophs involved in the fermentation and probably anaerobic oxidation of organic remains. Thus, the microbial ecosystems in Ediacaran ocean waters and sediments were more complex than previously thought, and these microbial processes controlled dynamic micro-environments in DST sediments where chert nodules were formed and fossils were mineralized. The results also show that variations in the relative abundances, activities, and interactions of co-existing microorganisms in DST sediments may have modulated δ13Corg shifts, causing local decoupling between δ13Corg and δ13Ccarb as measured in bulk samples.