Microbiological contribution to the formation of the manganese deposits in Sambe hot springs, Shimane, was investigated in combination with water chemistry, characterization of sediments and microbial community structure. Analysis of bacterial and fungal community structure based on DNA extracted from a Mn-oxidizing enrichment culture indicated close matches with Pseudomonas putida, Phoma sp. and Plectosphaerella cucumerina, all Mn-oxidizing microorganisms. These sediments were poorly crystalline and formed at neutral pH values, which is characteristic of biogenic precipitates. The EPMA results demonstrated a positive correlation between Mn and Ba contents in well-crystalline Mn oxide grains. Substantial Ba contents were observed inside Mn oxide grains. These findings indicated that Ba contents in sediments are influenced by not only aqueous Ba 2+ concentrations but also crystallinity of biogenic birnessite. Barium would be incorporated in birnessite during biomineralization.