Inventories of biodiversity are crucial for helping support conservation efforts, yet the deep sea, which is the largest biome on earth remains vastly understudied. Recent advances in molecular detection methods offer alternative techniques for studying inaccessible ecosystems, including those at depth. In this study we utilized environmental DNA metabarcoding, a first for studying deep-sea benthic environments in southern Africa, to assess the biological diversity and possible effect of trawling on these communities in the study area. Utilising sediment samples collected across a depth gradient and targeting a region of the cytochrome oxidase I (COI) gene, we recovered 444 OTUs across a wide array of species and genera, although many OTUs could only be assigned to higher taxonomic levels. Results showed that biodiversity differed significantly across depth, suggesting that even at relatively small spatial scales (~ 6 km) eDNA derived biodiversity detected variation linked to the depth gradient. No significant effects of trawling could be detected from eDNA analyses. Comparison of the OTU database with known species inventories from the sampled area revealed little overlap, highlighting the need for expanding barcoding efforts of deep-sea species to aid future eDNA survey efforts. Overall our results suggest that, with increased barcoding efforts, a combined approach of eDNA metabarcoding and physical sampling could capture a greater proportion of benthic deep-sea biodiversity. This provides provides additional opportunities to underpin conservation and management decision-making in the region, such as evaluating potential sites for future protection.