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Open pits, which remain after uranium (U) mining operations cease, can form meromictic lakes that develop suitable conditions for the containment of dissolved and colloidal metals. In this study, the distribution and speciation of U, nickel (Ni), and arsenic (As) in the water column of two meromictic pit lakes was investigated at the decommissioned Cluff Lake mine in northern Saskatchewan. The 28 m deep and older D pit had a chemocline at a 13 m depth, below which it turned anoxic and its meromixis was controlled by iron (Fe) cycling. Below the chemocline both Fe(III) and As(V) were reduced to Fe(II) and As(III), respectively. Iron cycling had a large effect on U distribution because reducing conditions prevented sulfide oxidation and a drop in pH in deeper layers. Metal-reducing bacteria were found to be present at, and below, the chemocline. In the deeper (90 m), larger and more recently flooded DJX pit, two chemoclines were observed at depths of 15 and 65 m. Both were linked to sharp U and Ni concentration gradients. Unlike the D pit, a transition to reducing conditions was not observed in the DJX pit’s water column. However, colloidal U, primarily associated with aluminum oxyhydroxides, was found below the first chemocline. Overall, the meromixis type determined the distribution and speciation of metals and bacteria in the investigated pit lakes, thus providing insights into the use of pit lakes as a potential bioremediation strategy.