Submarine mud volcanoes (MVs) are a type of cold seep environment where sediment, hydrocarbons and other reduced compounds are channelled upwards to the seafloor from significant depth. These sites can be ecological hotspots because of the potential microbial substrates present in the MV ejecta, and are a potential habitat for deep-sourced prokaryotes (Bacteria and Archaea). The microbial communities in sub-surface sediments from four separate MVs in the Gulf of Cadiz (Capt. Arutyunov, Bonjardim, Meknes and Porto) were investigated using a multidisciplinary approach. This involved cultivation and culture-independent molecular genetic-based methods, supplemented by basic pore water geochemistry, activity measurements and direct cell counts. Cultivation-independent 16S rRNA and functional (mcrA and dsrA) gene analyses revealed that the prokaryotes present in the MV sediments were often most closely related to uncultivated organisms. Phylogenetic groups representing major components of the sediment community in these sites included ANME-2a, ANME-la, Miscellaneous Crenarchaeota Group, Deltaproteobacteria and the JS1 candidate division, though community composition varied significantly between MV samples and with depth. Variation in community composition with depth through MV craters paralleled changes in pore water geochemistry indicating this is an important parameter influencing prokaryotic distribution in MV sediments. While containing certain phylogenetic groups 'characteristic' of the deep biosphere, the MV sediments also contained groups commonly associated with near-surface seep environments, suggesting the mud breccia had become colonised by organisms adapted to the present in situ conditions over time. Cultivation analysis showed novel organisms and important functional groups (methanogens and sulphate-reducers) could be cultivated from MV sediment. Pure cultures obtained from Capt. Arutyunov included a putative new species of Arcobacter named "Candidates Arcobacter subtericola" and species belonging to the genera Pseudomonas, Marinobacter, and Halomonas. Enrichments from Meknes contained Bacteria from the groups Bacteroidetes, Fusobacteria, Firmicutes, Spirochaetes, Desulfobulbaceae and Desulfovibrio, and Archaea belonging to the genera Methanogenium and Methanococcoides.