Calcitic speleothems in caves can form through abiogenic or biogenic processes, or through a combination of both. Many issues conspire to make the assessment of biogenicity difficult, especially when focusing on old speleothem deposits. This study reports on a multiproxy analysis of a Siberian stalactite, combining high-resolution microscopy, isotope geochemistry and microbially enhanced mineral precipitation laboratory experiments. The contact between growth layers in a stalactite exhibits a biogenic isotopic signature; coupled with morphological evidence, this supports a microbial origin of calcite crystals. SIMS δ13C data suggest that microbially mediated speleothem formation occurred repeatedly at short intervals before abiotic precipitation took over. The studied stalactite also contains iron and manganese oxides that have been mediated by microbial activity through extracellular polymeric substance (EPS)-influenced organomineralization processes. The latter reflect paleoenvironmental changes that occurred more than 500 000 yr ago, possibly related to the presence of a peat bog above the cave at that time. Microbial activity can initiate calcite deposition in the aphotic zone of caves before inorganic precipitation of speleothem carbonates. This study highlights the importance of microbially induced fractionation that can result in large negative δ13C excursions. The microscale biogeochemical processes imply that microbial activity has only negligible effects on the bulk δ13C signature in speleothems, which is more strongly affected by CO2 degassing and the host rock signature.