The emergence and diversification of eukaryotes during the Proterozoic is one of the most fundamental evolutionary developments in Earth’s history. The ca. 1-billion-year-old Lakhanda Lagerstätte (Siberia, Russia) contains a wealth of eukaryotic body fossils and offers an important glimpse into their ecosystem. Seeking to complement the paleontological record of this remarkable lagerstätte, we here explored information encoded within sedimentary organic matter (total organic carbon = 0.01–1.27 wt.%). Major emphasis was placed on sedimentary hydrocarbons preserved within bitumens and kerogens, including molecular fossils (or organic biomarkers) that are specific to bacteria and eukaryotes (i.e. hopanes and regular steranes, respectively). Programmed pyrolysis and molecular organic geochemistry suggest that the organic matter in the analyzed samples is about peak oil window maturity and thus sufficiently well preserved for detailed molecular fossil studies that include hopanes and steranes. Together with petrographic evidence as well as compositional similarities of the bitumens and corresponding kerogens, the consistency of different independent maturity parameters establishes that sedimentary hydrocarbons are indigenous and syngenetic to the host rock. The possible presence of trace amounts of hopanes and absence of steranes in samples that are sufficiently well preserved to retain both types of compounds evidences an environment dominated by anaerobic bacteria with no or very little inputs by eukaryotes. In concert with the paleontological record of the Lakhanda Lagerstätte, our study adds to the view that eukaryotes were present but not significant in Mesoproterozoic ecosystems.