This study aimed to isolate essential urease-producing bacteria required for biocementation using a down-flow hanging sponge (DHS) reactor and an isolation chip (ichip). As a result, almost all urea was decomposed to ammonium in the DHS reactor. Additionally, microbial community structure analysis based on 16S rRNA gene sequencing revealed the presence of various urease-producing bacteria in the DHS reactor. The isolated microorganisms not only possessed urease activity but also exhibited urea decomposition rates comparable to those of S. pasteurii. Analysis of the 16S rRNA gene of the isolated microorganisms showed high genetic similarity to S. pasteurii. Furthermore, the Sporosarcina genus and Bacillus genus accounted for only 1% of the microbial community structure. This suggests that combining DHS reactors with the ichip method allows for the isolation and cultivation of microorganisms with low abundance within the DHS reactors.