Recent technical developments have enabled the application of compound-specific isotope analysis (CSIA) of low (parts per billion) concentrations of 1,4-dioxane that are often found in groundwater at 1,4-dioxane-contaminated sites. However, to quantify 1,4-dioxane biodegradation, isotopic enrichment factors are needed to interpret the CSIA data obtained from field samples. In this study, the carbon and hydrogen isotopic enrichment factors (εC and εH, respectively) for 1,4-dioxane biodegradation have been determined for axenic propane- or isobutane-grown cultures of Rhodococcus rhodochrous ATCC 21198 and for tetrahydrofuran-grown cultures of Pseudonocardia tetrahydrofuranoxidans K1. The enrichment factors for propane-grown (εC = −2.7 ± 0.3‰, and εH = −21 ± 2‰) and isobutane-grown (εC = −2.5 ± 0.3‰, and εH = −28 ± 6‰) cells of strain 21198 were similar and substantially smaller than those determined for tetrahydrofuran-grown cells of strain K1 (εC = −4.7 ± 0.9‰, and εH = −147 ± 22‰). The presence of 1-butyne consistently inhibited both the biodegradation and isotopic fractionation of 1,4-dioxane, and this effect implicates monooxygenase enzymes in both the biodegradation and isotopic enrichment of 1,4-dioxane. Our results confirm that an increasing level of enrichment of heavier isotopes of carbon and hydrogen can be used to quantify 1,4-dioxane biodegradation and suggest CSIA can discriminate between the activities of monooxygenase-expressing bacteria expected to be prevalent in engineered, gaseous alkane-stimulated 1,4-dioxane treatment systems and those that may involve microbial metabolism of 1,4-dioxane as a natural attenuation process.