Comprehensive reconstruction of changes in eukaryotic communities in the recent past is useful for determining the response of the local ecosystems to global changes during the Anthropocene. We used DNA barcoding technology to reconstruct the marine eukaryotic communities of Beppu Bay, the Seto Inland Sea, Japan, over the past 50 years based on a short sediment core. Highly vulnerable DNA fragments were preserved in the sediments, possibly due to seasonally euxinic conditions. Analysis of the 18S rRNA V9 gene region indicated the temporal variability in eukaryotic communities, which consisted mainly of dinoflagellates and diatoms, in response to changes in the nutrient regime. The dominant species in the dinoflagellate genus Alexandrium changed as the water temperature increased. In addition, enhanced contributions by terrestrial plants and mosses were detected in flood sediments. Our results suggest that DNA fragments can be used as a proxy for the paleoenvironmental and paleoecological conditions in Beppu Bay.