Microbial DNA-based monitoring is a promising tool for reservoir monitoring that has been used mainly for shale reservoir development. In this study, long-term microbial DNA-based monitoring was applied to the Sarukawa oil field, which has a complex reservoir structure with no practical simulation model available. Fluid samples were collected periodically from nine production wells and two injection wells from October 2019 to July 2021. DNA was extracted from the samples, and the microbial composition was analyzed by 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing and real-time polymerase chain reaction (PCR). Based on similarities between the microbial profiles, the samples were classified into seven clusters that corresponded closely to the original fluid type (i.e., injection or production fluid) and specific environment (e.g., geological strata or compartments). A comparative analysis of the microbial profiles suggested possible well connectivity and water breakthrough. These results demonstrate that microbial DNA-based monitoring can provide useful information for optimizing production processes (e.g., waterflooding) in mature oil fields.