Studies are limited on microbial-driven sulfurous species reduction coupled with anaerobic ammonium oxidation (Sammox) in a water environment. In this study, thiosulfate-driven Sammox bacteria from Ningbo Critical Zone Observatory (CZO) groundwater, Zhejiang Province, China were enriched and analyzed. The results showed that all three groundwater samples contained bacteria with sulfur reduction, sulfide oxidation-dependent denitrification, and nitrogen fixation abilities. At the genus level, Arcobacter, Flavobacterium, Hydrogenophaga, Gemmobacter, Pseudomonas, and Methylophilus have higher abundance in three samples. Proteobacteria accounted for a large proportion at the phylum level in all groundwater samples, and are often found in the sulfate reduction coupled with the ammonium oxidation process. Sammox can be performed by synergistic action of bacteria, which may convert ammonium to nitrite and thiosulfate to sulfide. Arcobacter was the dominant bacteria in the medium to remove nitrite and thiosulfate/sulfide. Sammox may also be performed by a single bacterium, belonging to Planctomycetes, Verrucomicrobia, and Chlamydiae superphylum. Overall, bacterial-mediated Sammox exists in the environment of CZO groundwater.