The Korean Demilitarized Zone (DMZ) was established in 1953 during the cessation of the Korean War, which divided the Korean Peninsula into North and South Korea. The DMZ is a representative biodiversity resource because it limits human activities. The current status of faunal diversity in adjacent areas of the DMZ was investigated in five regions at 91 sites from 2015 to 2019. A total of 19,562 individuals were collected and identified, including 81 species, 19 families, and 11 orders. Zacco koreanus was the most abundant fish species (relative abundance of 21.9%), while other key species were Zacco platypus (18.2%), Rhynchocypris oxycephalus (5.2%), Rhynchocypris steindachneri (4.9%), and Pungtungia herzi (3.7%). Notably, twenty-nige endemic and seven Red Data Book species were recorded, and three exotic species, Carassius cuvieri, Micropterus salmoides, and Lepomis macrochirus were found at five sites. Human activities and disturbances were found at 34.0% of all sampling sites, and the highest disturbance factors were river dredging and agricultural use (seven sites), sewage (five sites), and fisheries and development (two sites). We recommend that the governments of South and North Korea collaboratively manage the DMZ to protect ecological diversity and maintain its status as a symbol of peace.