Twin Lakes, located on the Confederated Tribes of the Colville Indian Reservation in eastern Washington, USA, include North Twin Lake (NT) and South Twin Lake (ST). The mesotrophic, dimictic lakes are important recreational fishing sites for both warm-water bass and cold-water trout. To improve summertime cold-water habitat for trout in NT, dissolved oxygen (DO) addition to the hypolimnion, using liquid oxygen as an oxygen gas source, started in 2009. This study assessed mercury (Hg) in the water column, zooplankton and fish, and related water quality parameters, in Twin Lakes from 2009 to 2012. Because methylmercury (MeHg) buildup in lake bottom water is commonly associated with hypolimnetic anoxia, hypolimnetic oxygenation was hypothesized to reduce Hg in bottom waters and biota in NT relative to ST. Oxygen addition led to significantly higher DO (mean hypolimnetic DO: 2–8 mg/L versus b1 mg/L) and lower MeHg (peak mean hypolimnetic MeHg: 0.05–0.2 ng/L versus 0.15–0.4 ng/L) in North Twin. In North Twin, years with higher DO (2009 and 2011) exhibited lower MeHg in bottom waters and lower total Hg in zooplankton, inferring a positive linkage between oxygen addition and lower bioaccumulation. However, when comparing between the two lakes, Hg levels were significantly higher in zooplankton (total Hg range: 100–200 versus 50–100 μg/kg dry weight) and trout (spring 2010 stocking cohort of eastern brook trout mean total Hg: 74.9 versus 49.9 μg/kg wet weight) in NT relative to ST. Lower Hg bioaccumulation in ST compared to NT may be related to bloom dilution in chlorophyll-rich bottom waters, a vertical disconnect between the location of zooplankton and MeHg in the water column, and high binding affinity between sulfide and MeHg in bottom waters.