The fecal indicator bacterial species Escherichia coli is an important measure of water quality and a leading cause of impaired surface waters. We investigated the impact of the filter-feeding metazooplankton Daphnia magna on the inactivation of E. coli. The E. coli clearance rates of these daphnids were calculated from a series of batch experiments conducted under variable environmental conditions. Batch system experiments of 24 to 48 h in duration were completed to test the impacts of bacterial concentration, organism density, temperature, and water type. The maximum clearance rate for adult D. magna organisms was 2 ml h−1 organism−1. Less than 5% of E. coli removed from water by daphnids was recoverable from excretions. Sorption of E. coli on daphnid carapaces was not observed. As a comparison, the clearance rates of the freshwater rotifer Branchionus calyciflorus were also calculated for select conditions. The maximum clearance rate for B. calyciflorus was 6 × 10−4 ml h−1 organism−1. This research furthers our understanding of the impacts of metazooplankton predation on E. coli inactivation and the effects of environmental variables on filter feeding. Based on our results, metazooplankton can play an important role in the reduction of E. coli in natural treatment systems under environmentally relevant conditions. IMPORTANCE Escherichia coli is a fecal indicator bacterial species monitored by the U.S. Environmental Protection Agency to assess microbial water quality. Due to the potential human health implications linked to high levels of E. coli, it is important to understand the inactivation or reduction mechanisms in surface waters. Our research examines the capacities of two types of widespread filter-feeding freshwater metazooplankton, Daphnia magna and Brachionus calyciflorus, to reduce E. coli concentrations. We examine the impacts of different environmentally relevant conditions on the clearance rates. Our results contribute to a better understanding of the importance of metazooplankton in controlling E. coli concentrations and what conditions will reduce or increase grazing. These results provide baseline data to support future efforts to develop a quantitative model relating zooplankton uptake rates to relevant environmental variables.