Zooplankton communities in Boreal Shield lakes of south-central Ontario, Canada, have become increasingly exposed to the effects of multiple anthropogenic stressors, such as declines in calcium (Ca) and total phosphorus (TP) concentrations, shifts in predation regimes, and climate warming. The paleolimnological approach provides an effective means of examining cladoceran zooplankton communities prior to the onset of these major environmental stressors and assessing how the increasing impacts of these stressors have affected zooplankton community composition. We examined the chitinized remains of cladocerans from recent and pre-industrial (pre- ~1850s) sediments in 42 oligotrophic lakes from south-central Ontario and compared these assemblages using ANOSIM and SIMPER. Differences in cladoceran assemblages since pre-industrial times were related to five environmental variables that significantly influence cladoceran community composition in surface sediments. These included measured physical (depth), chemical [Ca, pH, sulphate, dissolved organic carbon (DOC)], and biological (fish community biomass) limnological variables. Two changes were recorded in the cladoceran species assemblages of the study lakes. The first was a significant increase in the ratio of pelagic species compared to littoral species over time in most lakes which we cannot attribute to any measured environmental variable with certainty but it likely represents a multiple stressor effect. The second was changes in relative abundances of daphniid cladocerans in several lakes since pre-industrial times, which we attribute to the interactive effects of several environmental stressors, including: differences in Ca availability within our lakes, resulting in decreased abundances of daphniids over time in lakes with lower Ca levels; long-term increases in DOC concentrations, which may provide refuge for daphniids from visual predators; and long-term declines in TP concentrations which may contribute to the dominance of species that are more efficient grazers, such as daphniids. Overall, this study provides field-based evidence that the modern-day cladoceran communities in south-central Ontario lakes are different than they were prior to human settlement in the region, and therefore these paleolimnological data provide a long-term, historical component to contemporary cladoceran datasets and an extended perspective on how multiple environmental stressors have impacted aquatic organisms from Boreal Shield lakes.