This study characterizes the effects of water–soil flooding volume ratio and flooding time on copper (Cu) desorption and toxicity following multiple floodings of field-collected soils from agricultural sites acquired under the Comprehensive Everglades Restoration Plan (CERP) in south Florida. Soils from four field sites were flooded with three water–soil ratios (2, 4, and 6 [water] to 1 [soil]) and held for 14 days to characterize the effects of volume ratio and flooding duration on Cu desorption (volume ratio and flooding duration study). Desorption of Cu was also characterized by flooding soils four times from seven field sites with a volume ratio of 2 (water) to 1 (soil) (multiple flooding study). Acute toxicity tests were also conducted using overlying waters from the first flooding event to characterize the effects of Cu on the survival of fathead minnows (Pimephales promelas), cladocerans (Daphnia magna), amphipods (Hyalella azteca), midges (Chironomus tentans), duckweed (Lemna minor), and Florida apple snails (Pomacea paludosa). Acute tests were also conducted with D. magna exposed to overlying water from the second and third flooding events. Results indicate that dissolved Cu concentrations in overlying water increased with flooding duration and decreased with volume ratio. In the multiple flooding study, initial Cu concentrations in soils ranged from 5 to 223 mg/kg (dw) and were similar to Cu concentration after four flooding events, indicating retention of Cu in soils. Copper desorption was dependent on soil Cu content and soil characteristics. Total Cu concentration in overlying water (Cuw) was a function of dissolved organic carbon (DOC), alkalinity, and soil Cu concentration (Cus): log(Cuw) = 1.2909 + 0.0279 (DOC) + 0.0026 (Cus) − 0.0038 (alkalinity). The model was validated and highly predictive. Most of the desorbed Cu in the water column complexed with organic matter in the soils and accounted for 99% of the total dissolved Cu. Although total dissolved Cu concentrations in overlying water did not significantly decrease with number of flooding events, concentrations of free Cu2+ increased with the number of flooding events, due to a decrease in DOC concentrations. The fraction of bioavailable Cu species (Cu2+, CuOH+, CuCO3) was also less than 1% of the total Cu. Overlying water from the first flooding event was only acutely toxic to the Florida apple snail from one site. However, overlying water from the third flooding of six out of seven soils was acutely toxic to D. magna. The decrease in DOC concentrations and increase in bioavailable Cu2+ species may explain the changes in acute toxicity to D. magna. Results of this study reveal potential for high Cu bioavailability (Cu2+) and toxicity to aquatic biota overtime in inundated agricultural lands acquired under the CERP.