A two‐step process, with anionic hydrolyzed polyacrylamide (HPAM) and cationic poly(vinylbenzyl trimethylammonium chloride) (PVBTMAC), was used to flocculate oil sands mature fine tailings (MFT). The charge density of HPAM and the dosages of HPAM and PVBTMAC were varied to maximize the dewatering of MFT and minimize the turbidity of the supernatant. The dual flocculant system performed better at lower doses than when used alone. The best HPAM charge density for fast initial settling rate and high sediment solids content was 32%, but it increased to 38% for best sediment dewatering, as measured by capillary suction time. The clearest supernatants were found when the zeta potential approached 0 mV because the cationic PVBTMAC neutralized the surface charges of the particles suspended in the MFT. More importantly, undiluted MFT was flocculated with the HPAM/PVBTMAC system and then filtered at low‐pressure (69 kPag), resulting in a filter cake with a high solids content of 61 wt.%. This study shows that treating MFT with a combination of anionic and cationic polymer is a flexible way to optimize flocculation performance.