Raman and Fourier-transform infrared (FTIR) microspectroscopic analyses were performed to examine the chemical signatures and their spatial distribution of organic-walled microfossils together with organic matter (OM) in a black chert nodule from the Ediacaran (635–551 Ma) Doushantuo Formation in China. Raman spectral parameters (I-1350/1600 values) reveal that the acritarchs, bundled filaments, and OM have different degrees of structural order, and that there is no significant difference in I-1350/1600 values among the different portions of individual acritarchs (i.e., inner structure, inner membrane, and outer wall). The IR mapping reveals portion-specific chemical signatures within the individual acritarch specimens. The inner membrane of Tianzhushania contains aliphatic C-H bonds. The acritarchs, including Tianzhushania, show chemically four types of inner structures: (1) aromatic C-H-rich type, (2) aliphatic C-H-rich type, (3) aromatic/aliphatic C-H-poor type, or (4) heterogeneous. All of the examined acritarchs have aromatic C-H rich outer wall, irrespective of the presence/absence of processes. These features support a eukaryotic cyst origin, although it is difficult to determine whether the acritarchs correspond to the cysts of Protista or Metazoa. The bundled filaments also consist of an aromatic C-H bond, but its origin is uncertain. The OM shows a high degree of variation in Raman spectral I-1350/1600 values, as does the IR spectral intensity ratio of asymmetric aliphatic CH3/CH2, alongside a heterogeneous spatial distribution of aromatic and aliphatic C-H bands. This suggests the contribution of various precursors derived from dead microbial cell debris and extracellular organic compounds. Overall, these results confirm the presence of diverse microorganisms in the Doushantuo microbiota. Furthermore, the spatial distribution of organic functional groups in individual microfossils could provide clues about the taxonomy of microfossils of unknown origin.