The Guaymas Basin (Gulf of California) hydrothermal vent area is known as a dynamic and hydrothermally vented sedimentary system, where the advection and production of a variety of different metabolic substrates support a high microbial diversity and activity in the seafloor. The main objective of our study was to explore the role of temperature and other environmental factors on community diversity, such as the presence of microbial mats and seafloor bathymetry within one hydrothermally vented field of 200 × 250 m dimension. In this field, temperature increased strongly with sediment depth reaching the known limit of life within a few decimeters. Potential sulfate reduction rate as a key community activity parameter was strongly affected by in situ temperature and sediment depth, declining from high rates of 1-5 μmol ml-1 d-1 at the surface to the detection limit below 5 cm sediment depth, despite the presence of sulfate and hydrocarbons. Automated Ribosomal Intergenic Spacer Analysis yielded a high-resolution fingerprint of the dominant members of the bacterial community. Our analyses showed strong temperature and sediment depth effects on bacterial cell abundance and Operational Taxonomic Units (OTUs) number, both declining by more than one order of magnitude below the top 5 cm of the sediment surface. Another fraction of the variation in diversity and community structure was explained by differences in the local bathymetry and spatial position within the vent field. Nevertheless, more than 80% of all detected OTUs were shared among the different temperature realms and sediment depths, after being classified as cold (T < 10°C), medium (10°C ≤ T < 40°C) or hot (T ≥ 40°C) temperature conditions, with significant OTU overlap with the richer surface communities. Overall, this indicates a high connectivity of benthic bacterial habitats in this dynamic and heterogeneous marine ecosystem influenced by strong hydrothermalism. © 2013 Meyer, Wegener, Lloyd, Teske, Boetius and Ramette.