This study investigates potential impacts connected to the removal of manganese nodules on deepsea sediment biogeochemistry and benthic microbial communities. The study site is located in the DISCOL Experimental Area (DEA) in the Peru Basin where a large scale disturbance experiment has been carried out in 1989. Investigations were performed during RV SONNE cruise SO242/2 in Sep. 2015 in the framework of the JPI Oceans programme ‘Ecological Aspects of deep sea mining’. The focus of the work presented here is on effects on the community structure, activity, and functions of benthic microorganisms. Sediment cores and manganese nodules were sampled in different disturbed and undisturbed areas. Overall, four microhabitats within the initial DISCOL disturbance tracks, a recent disturbance track created by an epibenthic sledge, and reference areas outside the DEA were investigated. Samples for laboratory incubations and analyses were taken with ROV and multicorer while fluxes were studied in situ with autonomous benthic chamber and micro profiler systems. Sediments were characterized by their colour, phytopigments, particulate carbon and nitrogen contents. The microbial activities were evaluated on the basis of diffusive oxygen fluxes, extracellular enzymatic activities, and uptake of radiolabelled compounds (i.e. bicarbonate and leucine). Microbial communities and diversities were characterized via 16S rRNA Illumina tag sequencing. Our data show that areas, where the simulated mining activity removed the reactive surface layer were most affected, even after 26 years. Most microbial and biogeochemical characteristics at these sites still resemble conditions of deeper sediment layers, with strongly reduced microbial abundances, activities of extracellular enzymes involved in organic matter degradation, respiration rates, and inorganic carbon fixation rates. A significant impact was also observed for the bacterial and archaeal community structure and diversity that differed considerably from communities found at reference sites. Similar effects were also identified at another microhabitat, where surface sediments were still present but largely reworked into ripple-like structures. Also here microbial and biogeochemical characteristics were more similar to subsurface layers than to undisturbed sites. We conclude that deep-sea mining reduces microbial activities, and changes their community structure and consequently their function in manganese nodule ecosystems for decades. Differences between microhabitats indicate that strong sediment redistribution and especially the loss of the surface layer represent the strongest impacts. Since microbes represent the basis of a multi-trophic food web, higher trophic levels and larger organisms may also be indirectly affected by the observed changes.