Abstract <p>Due to the lack of unambiguous fossils, the biogenicity of early Precambrian microbialites have often been questioned. To address this challenging issue, we conducted petrography, FESEM-EDX, Raman spectroscopy, and stable carbon isotope investigations on the carbonaceous laminae of hemispheroidal microbialites from the Paleoproterozoic carbonate deposits of Bajna Dolomite, Bijawar Group, India. Thin-section petrographic analysis reveals that the carbonate is mainly composed of dolomite, with alternating laminae rich in carbonaceous material associated with early diagenetic silica deposition. The carbonaceous matter is present in the form of sheaths and fragments dispersed in a three-dimensional orientation. The FESEM-EDX investigation of carbonaceous matter and the surrounding matrix demonstrates that the carbonaceous matter contains significant enrichment of cations such as Ca, Mg, K, Na, Al, and Fe, which are rare in the matrix of the immediate vicinity. This association of cations with the carbonaceous matter indicates the possibility that these carbonaceous matters are the remains of microbial communities predominantly comprising extracellular polymeric substance-secreting bacteria and cyanobacteria. The results of&#xa0;Raman spectroscopic analysis reveal minor spatial variations in thermal maturation and the corresponding maximum and minimum organic carbon disorder. These data reasonably indicate a biogenic origin for the carbonaceous material and the absence of any younger organic contamination.</p> Research highlights <p><UnorderedList Mark="Bullet"> <ItemContent> <p>Multi-proxy analyses confirm biogenic carbon in Paleoproterozoic microbialites.</p> </ItemContent> <ItemContent> <p>Cation-rich carbonaceous sheaths suggest EPS-producing microbes and cyanobacteria.</p> </ItemContent> <ItemContent> <p>Raman spectra exclude contamination and support syngenetic microbial origin.</p> </ItemContent> </UnorderedList></p>