Acid deposition has declined across eastern North America and northern Europe due to reduced emissions of sulfur and nitrogen oxides. Ecosystem recovery has been slow with limited improvement in surface water chemistry. Delayed recovery has encouraged acid-neutralization strategies to accelerate recovery of impaired biological communities. Lime application has been shown to increase pH and dissolved organic carbon (DOC), which could also drive increased mobilization of mercury (Hg) to surface waters. A four-year study was conducted within Honnedaga Lake’s watershed in the Adirondack region of New York to compare the effects of watershed and direct channel lime additions on Hg in stream water and macroinvertebrates. All treatments sharply increased stream pH and DOC concentrations, but large differences in the duration of impacts were apparent. The watershed treatment resulted in multi-year increases in concentrations and loads of total Hg (150%; 390%), DOC (190%; 350%) and nutrients, whereas total Hg and DOC increased for short periods (72–96 h) after channel treatments. No response of Hg in macroinvertebrates was evident following the watershed treatment, but a potential short-term and spatially constrained increase occurred after the channel treatment. Our observations indicate that both treatment approaches mobilize Hg, but that direct channel liming mobilizes considerably less than watershed liming over any period longer than a few days. During the final study year, increased methyl Hg concentrations were observed across reference and treated streams, which may reflect an extended dry period, highlighting that climate variation may also affect Hg dynamics.