Circulating drilling fluid is often regarded as a contamination source in investigations of subsurface microbiology. However, it also provides an opportunity to sample geological fluids at depth and to study contained microbial communities. During our study of deep subsurface microbiology of the Chinese Continental Scientific Deep drilling project, we collected 6 drilling fluid samples from a borehole from 2290 to 3350 m below the land surface. Microbial communities in these samples were characterized with cultivation-dependent and -independent techniques. Characterization of 16S rRNA genes indicated that the bacterial clone sequences related to Firmicutes became progressively dominant with increasing depth. Most sequences were related to anaerobic, thermophilic, halophilic or alkaliphilic bacteria. These habitats were consistent with the measured geochemical characteristics of the drilling fluids that have incorporated geological fluids and partly reflected the in-situ conditions. Several clone types were closely related to Thermoanaerobacter ethanolicus, Caldicellulosiruptor lactoaceticus, and Anaerobranca gottschalkii, an anaerobic metal-reducer, an extreme thermophile, and an anaerobic chemoorganotroph, respectively, with an optimal growth temperature of 50–68°C. Seven anaerobic, thermophilic Fe(III)-reducing bacterial isolates were obtained and they were capable of reducing iron oxide and clay minerals to produce siderite, vivianite, and illite. The archaeal diversity was low. Most archaeal sequences were not related to any known cultivated species, but rather to environmental clone sequences recovered from subsurface environments. We infer that the detected microbes were derived from geological fluids at depth and their growth habitats reflected the deep subsurface conditions. These findings have important implications for microbial survival and their ecological functions in the deep subsurface.