The stratigraphic record of organic matter in the c. 1.97 Ga Zaonega Formation (ZF), Onega Basin, northwestern Russia, exhibits a distinct negative δ13C excursion (δ13Corg from −25 to −40‰ VPDB), which was previously interpreted either to reflect a disturbance in the global carbon cycle after the Great Oxidation Event, or to have been caused by an increase in basinal methanotrophic activity. In order to assess the nature of primary biomass and the effects of post-depositional alteration, we here report the sample-scale carbon isotopic characteristics of organic matter in two drill cores from the ZF, covering 500 m of stratigraphy, by using secondary ion mass spectrometry (SIMS). The results confirm that the organic matter has to a large extent preserved the primary isotopic signatures, whereas secondary effects are limited (<4‰). The sample-scale isotopic heterogeneity, defined as the difference between the maximum and minimum δ13C values obtained by SIMS from every individual sample, increases from typically <5‰ in the lower part of stratigraphy to systematically larger values (up to 11‰) in the upper part, which coincides with the decreasing trend of δ13Corg of bulk samples from −25 to −40‰. Samples with either relatively high (c. −25‰) or low (c. −40‰) δ13Corg values have small sample-scale isotopic heterogeneities, while samples with intermediate δ13Corg values (between −25 and −40‰) have significantly larger heterogeneities. These observations imply the co-existence of photoautotrophic and methanotrophic biomass during deposition of the upper part of the stratigraphy. Our study provides insight into the carbon isotopic characteristics of organic matter and suggests that the negative excursion of δ13Corg in the ZF is induced by a methanotrophic microbial ecosystem sustained by seepage of thermogenic methane during the deposition of the ZF and contemporaneous igneous activities.