In situ infrared (IR) and Raman microspectroscopy have been conducted on Neoproterozoic, organic-walled microfossils (prokaryotic fossils) in doubly polished, petrographic thin sections in order to detect their spectral signatures. The microfossils are very well preserved and occur in black chert from the approximately 850 million-year-old Bitter Springs Formation, Northern Territory, Australia. Raman microspectroscopy on two species of microfossils, one a filament and the other a coccoid, shows disordered peaks (D peak, 1340 cm(-1)) and graphite peaks (G peak, 1600 cm(-1)), indicating that they consist of disordered carbonaceous materials. IR micro-mapping results of the filament reveal that the distributions of peak heights at 2920 cm(-1) (aliphatic CH(2)), 1585 cm(-1) (aromatic C-C), and 1370 cm(-1) (aliphatic CH(3)) match the shape of the filamentous microfossil. These results suggest that IR microspectroscopy can be used for in situ characterization of organic polar signatures that morphologically indicate microfossils embedded in chert by using doubly-polished rock (petrographic) thin section samples. Further, these methods can be applied to controversial microfossil-like structures to test their biogenic nature.