Gulf of Mexico cold seeps characterized by variable compositions and magnitudes of hydrocarbon seepage were sampled in order to investigate the effects of natural oils, methane, and non-methane hydrocarbons on microbial activity, diversity, and distribution in seafloor sediments. Though some sediments were characterized by relatively high quantities of oil, which may be toxic to some microorganisms, high rates of sulfate reduction (SR, 27.9±14.7mmolm-2d-1), anaerobic oxidation of methane (AOM, 16.2±6.7mmolm-2d-1), and acetate oxidation (2.74±0.76mmolm-2d-1) were observed in radiotracer measurements. In many instances, the SR rate was higher than the AOM rate, indicating that non-methane hydrocarbons fueled SR. Analysis of 16S rRNA gene clone libraries revealed phylogenetically diverse communities that were dominated by phylotypes of sulfate-reducing bacteria (SRB) and anaerobic methanotrophs of the ANME-1 and ANME-2 varieties. Another group of archaea form a Gulf of Mexico-specific clade (GOM ARC2) that may be important in brine-influenced, oil-impacted sediments from deeper water. Additionally, species grouping within the uncultivated Deltaproteobacteria clades SEEP-SRB3 and -SRB4, as well as relatives of Desulfobacterium anilini, were observed in relatively higher abundance in the oil-impacted sediments, suggesting that these groups of SRB may be involved in or influenced by degradation of higher hydrocarbons or petroleum byproducts. © 2010 Elsevier Ltd.