A well preserved and diversified microfossil assemblage is reported from the Meso–Neoproterozoic Mbuji-Mayi Supergroup in the Kasai oriental Province, central part of Democratic Republic of Congo. A total of 49 taxa belonging to 27 genera were identified, including 11 species of unambiguous eukaryotes, 10 species of possible eukaryotes or prokaryotes and 28 species of probable bacteria. This assemblage is more diverse than previously reported but includes taxa reported in coeval worldwide assemblages. It is characterized by abundant sphaeromorphs, filamentous colonial aggregates and filamentous forms, as well as a relatively low diversity of acanthomorphs including the Late Mesoproterozoic and Early Neoproterozoic index fossil – Trachyhystrichosphaera aimika – reported for the first time in Central Africa. This species co-occurs with other taxa also reported for the first time in Africa: Trachyhystrichosphaera botula, Jacutianema solubila, cf. Tappania sp., Valeria elongata and numerous other taxa. Correlation with other geochronologically constrained successions that contain Trachyhystrichosphaera confirms T. aimika as promising index fossil to define the Late Mesoproterozoic–Early Neoproterozoic interval. The available biostratigraphic data enable to suggest a minimum Tonian age for the Mbuji-Mayi Supergroup. This age is consistent with the published and new geochronological data. Comparison with worldwide Proterozoic assemblages permits to define microfossil assemblages useful for biostratigraphy. This study significantly improves our understanding of the diversity of the Late Mesoproterozoic–Early Neoproterozoic biosphere, and in particular the diversification of early eukaryotes, preserved in the Democratic Republic of Congo rock record and more broadly in Africa where micropaleontological investigations are sparse.