Characterization of the biodegradability of herbicidal ionic liquids (HILs) using the industry standard activated sludge methodology is thought to be insufficient to fully understand the biodegradation of HILs in the environment because cations and anions of the HILs may have different potential for biodegradation in aquatic and terrestrial systems. To test this hypothesis, we conducted laboratory microcosm experiments using C-13-labeled cations and anions forming benzyldodecyldimethylammonium 4-chloro-2-methylphenoxyacetate ([C-12-BA][MCPA]) and evaluated their biodegradation potential in soil with a known history of herbicide exposure, and in water (mineral medium) augmented with microorganisms present in activated sludge. The biodegradability of the cation and anion was found to depend on the test system (water or soil). The cation was mineralized in water, whereas the anion was fully utilized in soil. These results suggest that performing biodegradation tests using differently C-13-labeled species of the target HIL and mimicking various environmental compartments (e.g., soil, activated sludge) is needed to provide a better understanding of the fate of HILs in the environment. They also indicate that biodegradation kinetic parameters of HILs derived from experiments performed in aqueous systems should not be used to estimate biodegradation rates in terrestrial environments.