Anthropogenic litter negatively impacts the marine environment and threatens biodiversity. At the same time, it represents a suitable substrate for the settlement of sessile species, thus potentially altering composition and structure of soft bottom benthic assemblages. By using a Remotely Operated Vehicle (ROV), we hereby investigated patterns of abundance, distribution, and origin of benthic litter in three subtidal habitats of a heavily urbanized area and tested whether litter distribution related to patterns of fish and mega- and macro-benthic diversity. Litter accumulation mostly occurred on soft bottoms, while rocky substrata were the least affected, albeit being particularly threatened by sea-based pollution. As expected, the highest biodiversity was observed on rocky bottoms, hosting notable biogenic formations (Cladocora caespitosa, Leptogorgia sarmentosa) despite the area is historically affected by anthropogenic activities. No correlation was found between biota and marine litter, suggesting that litter does not apparently influence biodiversity and distribution of the investigated assemblages.