Microbes are key players in marine sediments, yet they are not accessed routinely by monitoring programs. Here, we investigate the spatial and vertical trends in the abundance, activity and diversity of benthic archaea, bacteria and fungi of the southeastern Mediterranean Sea (SEMS), based on ∼150 samples collected by the National Monitoring Program in 2018-2020 in coastal, as well as deep-sea transects across the Israeli exclusive economic zone, using vertical profiles of short sediment cores (0-1, 1-2, 4-5, 9-10 and 19-20 cm below surface). Microbial abundance was usually low (0.01 ×108 to 0.21×108 cells gr-1 sediment), while heterotrophic productivity was the highest in the nearshore stations (12±4 ng C gr-1 sediment h-1), as opposed to 0.5±0.9 ng C gr-1 sediment h-1 at the offshore sites. Using amplicon sequencing of marker genes, we identified the changes in the diversity of microbes along environmental gradients, in the four dimensions (geographic location, seabed depth, distance from the sediment surface and time). We show high taxonomic diversity of bacteria and archaea (Shannon’s H’ 5.0-6.9) and lesser diversity of fungi (Shannon’s H’ 0.2-4.8). We use DESeq2 analyses to highlight the role of ammonia-oxidizing Nitrososphaeria in the aerated sediments of the continental slope and deep bathyal plain stations and organotrophic lineages in coastal, shelf, slope, and abyssal plain sediments. Based on taxonomic diversity, we infer the metabolic potential of these communities. Analyses of fungi diversity and guilds suggest the prevalence of the saprotrophic and pathotrophic microfungi Ascomycota (70±23%) and Basidiomycota (16±18%) in the SEMS sediments. We provide a comprehensive baseline of benthic microbial populations in the SEMS and pledge for the use of microbial indices in biomonitoring of the marine environment.