Oil sands tailings ponds contain large volumes (∼108 m3) of fine tailings, originating from bitumen production by surface mining. These sediment rapidly in dilute suspension but then form a network, which consolidates much more slowly. The overall process increases solid content to up to 85% (w/w) and is referred to as tailings densification. Addition of gypsum (CaSO4·2H2O) to a mixture of sand and fines gives a non-segregating, consolidated tailings slurry in which calcium ions serve as a cross-linking agent. Tailings ponds also harbor active anaerobic microbial consortia, which are thought to contribute to densification through microbial activity, including gas production, creating dewatering channels. To determine the roles of calcium ions and anaerobic microbial activity in tailings sedimentation, we placed 70% (v/v) tailings, containing 77% (w/w) solids, and 30% (v/v) defined medium with various amendments in anaerobic test tubes with an N2–CO2 headspace. Following mixing the initial sedimentation rate R of the water-tailings boundary and the final percentage (v/v) of sedimentation SF were measured. Amendment with 0–20 mM CaCl2 increased R from 0.006 to up to 0.012 day−1, but decreased SF from 14–15% to 8–10% (v/v), whereas subsequent amendment with lactate increased both R and SF. To determine the effect of the type of anaerobic microbial activity, tubes were amended with (i) 20 mM NaCl or 10 mM CaCl2, (ii) 10 mM Na2SO4 or 10 mM CaSO4, (iii) 20 mM NaNO3 or 10 mM Ca(NO3)2, or (iv) no additions. Following mixing, duplicate tubes were monitored continuously to determine SF, whereas another set of duplicate tubes was re-mixed once per week to determine R, as well as headspace methane, and the concentrations of sulfate, sulfide, nitrate and nitrite in the supernatant fluid. Microbial activity was boosted after 63 days by adding 20 mM lactate to all tubes. The data for this experiment also indicated that R increased, whereas SF decreased by addition of calcium ions. Lactate significantly boosted microbial activity with increased methanogenesis, sulfate reduction or nitrate reduction being observed in tubes amended with no electron acceptor, sulfate or nitrate, respectively. Addition of lactate increased SF by 2–4% (v/v) in most tubes, except in tubes with Ca(NO3)2 in which SF increased by 15% (v/v). The solids content increased from 69 to 78% under these conditions, representing a significant progression to the maximum values observed in tailings ponds over a short period of time.