Anthropogenic pressures can result in a competitive advantage for benthic macroalgae over reef-building corals. Information on natural variation of benthic algae over long timescales can help assess current degradation and predict future response to environmental change. However, long-term monitoring records of benthic algae do not pre-date the start of anthropogenic pressures. To address this gap, we present a 400-year record of benthic macroalgae occurrences recovered from ancient environmental DNA from the inshore central Great Barrier Reef (GBR). We extracted aeDNA from ten sediment cores dated using U/Th radiometric age-dating. This period includes the onset of European colonization when large-scale land-clearing substantially increased sedimentation and nutrient influx. Our 18S metabarcoding record contains macroalgae that are common on present-day inshore reefs and includes previously unrecorded taxa at these sites. The aeDNA macroalgae community reflects known local spatial patterns, but there are no large-scale community shifts or fluctuations through time, including before or after European colonization, consistent with the local fossil record of corals. At some sites macroalgal occurrences of Rhodophyta and Phaeophyceae increase at the onset of European colonization, but this response was not universal. Our study shows metabarcoding of aeDNA can be used to recover patterns in the distribution of macroalgae over centennial time scales.