The initial development and diversity of an in situ subsurface microbial community producing bacteriogenic iron oxides (BIOS) were investigated at the initiation of biofilm growth (2-month period) and after a 1-year period of undisturbed growth. Water chemistry data, samples of iron encrusted biofilm material and groundwater were collected from BRIC (BIOS reactor, in situ, continuous flow) apparatuses installed 297 m below sea level at the Äspö Hard Rock Laboratory (HRL) in south eastern Sweden. Comparisons between the BIOS BRIC system and an anaerobic control (AC) BRIC revealed that water mixing at the inflow leads to profuse development of BIOS related to a slightly elevated level of O2 (up to 0.3 mg L−1 at the transition zone between BIOS development and non-development) and elevated Eh (>120 mV) in the first 70 mm of water depth. Decreases in dissolved and particulate iron were connected to the visible appearance of BIOS biofilms. The basic phylogenetic diversity of this site was evaluated using amplified ribosomal DNA restriction enzyme analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE) and partial sequencing of 16S rDNA. From 67 clones that were positive for 16S rDNA inserts, a total of 42 different ARDRA profiles were recognized, representing four bacterial phyla and 14 different metabolic lifestyles. DGGE profiles indicated that there are differences in the representative bacteria when considering either BIOS biofilms or groundwater. DGGE also indicated that the DNA extraction protocols and any polymerase chain reaction biases were consistent. Bacterial metabolic groups associated with indirect metal adsorption and reduction along with bacteria utilizing many alternative electron acceptors were strongly represented within the clones. This study indicates that the microbial diversity of BIOS is greater than previously thought.