Marine sediments host abundant and diverse bacterial and archaeal assemblages that play a key role in global biogeochemical cycles. However, patterns of prokaryotic diversity and the environmental factors shaping microbial assemblage composition in coastal subsurface sediments remain poorly understood, particularly within the first meters below the seafloor. Here, we used 16S rRNA gene metabarcoding to investigate bacterial and archaeal diversity in subsurface sediments collected at 50, 100, and 200 cm below the seafloor from seven stations in the Gulf of Naples. The investigated sediment layers differed in grain size, redox conditions and organic matter content, while contamination levels generally decreased with sediment depth, reflecting temporal changes in industrial inputs from the adjacent coastal area. Bacterial sequences accounted for ca. 70% of total reads, with Dehalococcoidia representing the most abundant bacterial class, whereas archaeal sequences were mainly assigned to Bathyarchaeia. Prokaryotic diversity, expressed as Amplicon Sequence Variants (ASVs), was highest in the deepest sediment layer, where chemical contamination was lowest. More than 1,000 ASVs were exclusively detected in specific sediment layers, suggesting that specific geochemical conditions may contribute to select distinct microbial assemblages. At the same time, the different sediment layers shared several ASVs, accounting for ca. 31% of the total reads. Among these shared taxa, members affiliated with Lokiarchaeia were consistently detected, suggesting the occurrence of prokaryotic lineages with broad ecological tolerance and/or metabolic versatility under changing environmental conditions, even in highly contaminated subsurface sediments. Overall, these findings provide new insights into the vertical distribution of bacterial and archaeal diversity in coastal benthic ecosystems and suggest that subsurface microbial communities may represent sensitive indicators of environmental changes. By linking microbial diversity patterns to sediment geochemistry and contamination history, these results may contribute to improving ecological monitoring strategies and supporting restoration-oriented assessments in impacted coastal ecosystems.