1,2-Dichloroethane (1,2-DCA) is a widespread groundwater contaminant known to be biodegradable under aerobic conditions via enzymatic oxidation or hydrolytic dehalogenation reactions. Current literature reports that stable carbon isotope fractionation of 1,2-DCA during aerobic biodegradation is large and reproducible (−27 to −33‰). In this study, a significant variation in the magnitude of stable carbon isotope fractionation during aerobic biodegradation was observed. Biodegradation in experiments involving microcosms, enrichment cultures, and pure microbial cultures produced a consistent bimodal distribution of enrichment factors (ε) with one mean ε centered on −3.9 ± 0.6‰ and the other on −29.2 ± 1.9‰. Reevaluation of ε in terms of kinetic isotope effects 12k/13k gave values of 12k/13k = 1.01 and 1.06, which are typical of oxidation and hydrolytic dehalogenation (SN2) reactions, respectively. The bimodal distribution is therefore consistent with the microbial degradation of 1,2-DCA by two separate enzymatic pathways. This interpretation is further supported in this study by experiments with pure strains of Xanthobacter autotrophicus GJ10, Ancylobacter aquaticus AD20, and Pseudomonas sp. Strain DCA1 for which the enzymatic degradation pathways are well-known. A small fractionation of −3.0‰ was measured for 1,2-DCA degradation by Pseudomonas sp. Strain DCA1 (monooxygenase enzyme), while degradation by the hydrolytic dehalogenase enzyme by the other two pure strains was characterized by fractionation of −32.3‰.