Oil sands tailings ponds in Alberta (Canada) are strongly stratified ecosystems structured in an upper water layer and underlying mud layers that harbour a diversity of microorganisms, contributing to hydrocarbon degradation and elemental cycling. Until now not much is known about the biogeochemistry of the ponds and their spatial structure is not well explored yet. An understanding of microbial activity and community composition is important, in particular, in order to determine potential effects on pond properties and long term development. Therefore, the purpose of the present study was to identify reactive zones of iron, carbon and sulfur cycling in an active tailings pond, by comparing biogeochemical data along two depth profiles. For both profiles a zone of intense sulfur cycling was substantiated by maxima of: (a) dissolved and solid sulfides (b) sulfate reduction rates and thiosulfate oxidation potentials (c) viable counts of sulfate reducers and relative abundances of functional genes. In addition, methanogenesis and microbial iron reduction were shown to be important electron accepting processes in the ponds. All processes coexisted in a zone of intense elemental cycling at a depth of 1–4 m below the water-mud interface, where fresh tailings are likely to accumulate. Microbial activity and biomass decreased with depth, where tailings had higher age and density. While the upper mud layers were influenced by the presence of different archaea, the microbial communities showed an increased presence of bacterial species at depth. Insights from qPCR, 35S radiotracer technique and stable isotope analysis mirrored some differences between the profiles, regarding sulfur and carbon cycling. Despite this, both profiles showed remarkably similar patterns of microbial community composition and activity, revealing a good reproducibility of biogeochemical cycling within a few meters.