1,4-Dioxane (dioxane) is a potential carcinogen widely used as a stabilizer for chlorinated solvents, and it exhibits high mobility in groundwater. Dioxane is recalcitrant to biodegradation, and its physicochemical properties preclude effective removal by volatilization or adsorption. Through this long-term microcosm study, we have assessed the natural attenuation potential of dioxane for multiple sediment and groundwater samples collected from three sites located in Los Angeles, CA. Groundwater and sediment samples were taken from three locations at each plume, representing the source zone, middle and leading edge. A total of 13 monitoring wells were sampled to prepare the microcosms and subsequently assess the indigenous potential to biodegrade dioxane. The microcosms were spiked with 14C-labeed dioxane to assess mineralization potential (per 14CO2 recovery). No dioxane loss and less than 8% CO2 recovery was observed in the negative controls, indicating that dioxane removal (and mineralization) was due to biodegradation. Positive control microcosms amended with the dioxane degrader Pseudonocardia dioxivorans CB1190 exhibited dioxane degradation activity statistically indistinguishable from observed batch incubations prepared with mineral media, indicating an absence of inhibitory compounds in source zone samples. Complete dioxane removal, exhibiting linear (zero-order) kinetics (indicative of saturated enzymes), was observed during 24 weeks incubation in all biologically active, unaugmented microcosms. Up to 43% mineralization as CO2 and 5% to 7% biomass growth was observed in unaugmented microcosms experiencing rapid dioxane loss. Degradation activity decreased with increasing distance from the contaminant source zone, presumably due to less acclimation. Source-zone microcosms from Site 1 exhibited relatively high biodegradation activity (323.9 ± 7.6 µg/L/day) and were respiked with dioxane for confirmatory purposes. The respike (2 ppm dioxane) was degraded faster within four weeks, suggesting a higher level of acclimation (possibly due to the growth of indigenous dioxane degraders) after the initial 24 week study. Source-zone microcosms from Site 2 and 3 exhibited biodegradation activities of 1.4 ± 0.09 µg/L/day and 47.1 ± 1.8 µg/L/day, respectively. Overall, these results show that indigenous microorganisms capable of degrading dioxane are present at the three sites considered, and suggest that monitored natural attenuation should be considered as a remedial response.