1,4-Dioxane, a probable human carcinogen, is a co-contaminant at many chlorinated solvent-contaminated sites. Although numerous 1,4-dioxane-degrading aerobic bacteria have been isolated, almost no information exists on the microorganisms able to degrade this chemical under anaerobic conditions. Here, the potential for 1,4-dioxane biodegradation was examined using multiple inocula and electron acceptor amendments. The inocula included uncontaminated agricultural soils and river sediments as well as sediments from two 1,4-dioxane contaminated sites. Five separate experiments involved the examination of triplicate live microcosms and abiotic controls for approximately 1 year. Compound-specific isotope analysis (CSIA) was used to further investigate biodegradation in a subset of the microcosms. Also, DNA was extracted from microcosms exhibiting 1,4-dioxane biodegradation for microbial community analysis using 16S rRNA gene amplicon high-throughput sequencing. Given the long incubation periods, it is likely that electron acceptor depletion occurred and methanogenic conditions eventually dominated. The iron/EDTA/humic acid or sulfate amendments did not result in 1,4-dioxane biodegradation in the majority of cases. 1,4-dioxane biodegradation was most commonly observed in the nitrate amended and no electron acceptor treatments. Notably, both contaminated site sediments illustrated removal in the samples compared to the abiotic controls in the no electron acceptor treatment. However, it is important to note that the degradation was slow (with concentration reductions occurring over approximately 1 year). In two of the three cases examined, CSIA provided additional evidence for 1,4-dioxane biodegradation. In one case, the reduction in 1,4-dioxane in the samples comparing the controls was likely too low for the method to detect a significant C-13/C-12 enrichment. Further research is required to determine the value of measuring H-2/H-1 for generating evidence for the biodegradation of this chemical. The microbial community analysis indicated that the phylotypes unclassified Comamonadaceae and 3 genus incertae sedis were more abundant in 1,4-dioxane-degrading microcosms compared to the live controls (no 1,4-dioxane) in microcosms inoculated with contaminated and uncontaminated sediment, respectively. The relative abundance of known 1,4-dioxane degraders was also investigated at the genus level. The soil microcosms were dominated primarily by Rhodanobacter with lower relative abundance values for Pseudomonas, Mycobacterium, and Acinetobacter. The sediment communities were dominated by Pseudomonas and Rhodanobacter. Overall, the current study indicates 1,4-dioxane biodegradation under anaerobic and, likely methanogenic conditions, is feasible. Therefore, natural attenuation may be an appropriate cleanup technology at sites where time is not a limitation.