Benthic diatoms are sensitive indicators of environmental conditions at the seabed. In this study, benthic diatom communities at two brine outfall sites of reverse osmosis (RO) seawater desalination plants in Larnaca and Dhekelia, Cyprus, were investigated using a classical, microscopy-based approach and environmental DNA metabarcoding. In general, the diversity of diatoms measured by both methods (microscopy and eDNA metabarcoding), increased by distance from the brine discharge. Increased TOC and nutrient enrichment at brine outfalls contributed to decreased diatom diversity at the Larnaca outfalls, but the diatom diversity at Dhekelia was not driven by abiotic factors. The diatom communities at the outfalls were shown to be distinct and showed temporal variation across the sampling seasons with eDNA metabarcoding, but this was the case only for Dhekelia with microscopy. The results highlight the effect of local biogeography and different brine mixing methods on diatom diversity. The results revealed that conventional morphological methods and eDNA metabarcoding rarely leads to similar conclusions. However, the complementary results emphasise that more information can be derived when combining the methods for biodiversity impact assessments.