Abstract The bacterial diversity in a diffuse flow hydrothermal vent habitat at Axial Volcano, Juan de Fuca Ridge was examined shortly after an eruptive event in 1998 and again in 1999 and 2000 using PCR-amplified 16S rRNA gene sequence analyses. While considerable overlap with deep-sea background seawater was found within the alpha- and gamma-proteobacteria, unique subseafloor phylotypes were distinguishable. These included diverse members of the epsilon-proteobacteria, high temperature groups such as Desulfurobacterium, Gram-positive bacteria, and members of novel candidate divisions WS6 and ABY1. Phylotype richness was highest in the particle-attached populations from all three sampling periods, and diversity appeared to increase over that time, particularly among the epsilon-proteobacteria. A preliminary model of the subseafloor is presented that relates microbial diversity to temperature, chemical characteristics of diffuse flow fluids and the degree of mixing with seawater.