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Representative biomarkers (e.g., n-alkanes), diversity and microbial community in the aquifers contaminated by high concentration of arsenic (As) in different sediment depth (0–30 m) in Jianghan Plain, Hubei, China, were analyzed to investigate the potential mechanism of As enrichment in groundwater. The concentration of As was abundant in top soil and sand, but not in clay. The analysis of the distribution of n-alkanes, CPI values, and wax to total n-alkane ratio (Wax(n)%) indicated that the organic matter (OM) from fresh terrestrial plants were abundant in the shallow sediment. However, n-alkanes have suffered from significant biodegradation from the depth of 16 m to 30 m. The deposition of fresh terrestrial derived organic matters may facilitate the release of As from sediment to groundwater in the sediment of 0–16 m. However, the petroleum derived organic matters may do the favor to the release of As in the deeper section of borehole (16 m to 30 m). The 16S rRNA gene sequences identification indicated that Acidobacteria, Actinomycetes and Hydrogenophaga are abundant in the sediments with high arsenic. Therefore, microbes and organic matters from different sources may play important roles in arsenic mobilization in the aquifers of the study area.