To examine microbe-mineral interactions in subsurface oceanic crust, we evaluated microbial colonization on crustal minerals that were incubated in borehole fluids for 1 year at the seafloor wellhead of a crustal borehole observatory (IODP Hole U1301A, Juan de Fuca Ridge flank) as compared to an experiment that was not exposed to subsurface crustal fluids (at nearby IODP Hole U1301B). In comparison to previous studies at these same sites, this approach allowed assessment of the effects of temperature, fluid chemistry, and/or mineralogy on colonization patterns of different mineral substrates, and an opportunity to verify the approach of deploying colonization experiments at an observatory wellhead at the seafloor instead of within the borehole. The Hole U1301B deployment did not have biofilm growth, based on microscopy and DNA extraction, thereby confirming the integrity of the colonization design against bottom seawater intrusion. In contrast, the Hole U1301A deployment supported biofilms dominated by Epsilonproteobacteria (43.5% of 370 16S rRNA gene clone sequences) and Gammaproteobacteria (29.3%). Sequence analysis revealed overlap in microbial communities between different minerals incubated at the Hole U1301A wellhead, indicating that mineralogy did not separate biofilm structure within the 1-year colonization experiment. Differences in the Hole U1301A wellhead biofilm community composition relative to previous studies from within the borehole using similar mineral substrates suggest that temperature and the diffusion of dissolved oxygen through plastic components influenced the mineral colonization experiments positioned at the wellhead. This highlights the capacity of low abundance crustal fluid taxa to rapidly establish communities on diverse mineral substrates under changing environmental conditions such as from temperature and oxygen.