To clarify the effects of intertidal groundwater discharge on macrofaunal community structures of tidal flats, the species richness, abundance and biomass of macrobenthos at the groundwater-seepage area were assessed quantitatively, measuring environmental conditions including sediment temperature, median diameter, ORP and ignition loss, and salinity, water temperature and nutrient concentration of pore water at four sites (Nakatsu River mouth, Sanbyakuken, Higashihama, Oshinden) on the Nakatsu tidal flat, Oita prefecture, Japan, at low tides in June and July, 2019. A total of 1,233 individuals, belonging to 51 macrobenthic species (37 families) were collected. Mean species richness and biomass were significantly greatest at Higashihama, whereas mean abundance was greatest at the Nakatsu River mouth. Lower species richness, abundance and biomass were recorded at Sanbyakuken. At Higashihama, infaunal species such as the bivalve Glauconome angulata and polychaete Ceratonereis erythraeensis were dominant in the lower salinity and higher nutrient environments. A canonical correspondence analysis showed a positive correlation between nutrients and dominant bivalve abundance. The overall results suggested that intertidal groundwater influences the macrofaunal community structure, providing low salinity and nutrient-rich conditions, which contribute to the high productivity and diversity of marine organisms on tidal flats.