Anoxic subseafloor crustal fluids (60-65 °C and pH 7.0-7.3) from Juan de Fuca Ridge (JdFR) flank were used to enrich and potentially isolate anaerobic chemosynthetic microorganisms driving primary production in these environments. Selective enrichments using H2 as the energy source during the anaerobic respiration (reduction) of CO2 or NO3- for the fixation of CO2 during primary production were incubated at temperatures between 65 °C and 80 °C. Two successful CO2 reducing cultures were obtained after six consecutive serial dilutions at 75 °C, and three NO3- reducing cultures were obtained after five consecutive serial dilutions at 65 °C or 75 °C. Cellular morphologies and sizes tend to vary between cultures, with CO2 reducing cultures growing as small cocci capable of biofilm formation and NO3- reducing cultures growing as small cocci that later develop into elongated filamentous cells and aggregates.  Despite the higher free energy of reaction generally associated with the microbial oxidation of H2 via NO3- reduction, in comparison to the microbial oxidation of H2 via CO2 reduction, CO2 reducing cultures showed significantly faster doubling times while also generally reaching higher cell densities in vitro. Observed growth differences between the two CO2 reducing cultures and three NO3- reducing cultures could indicate a broader lifestyle adaptation to low NO3- concentrations in the highly reduced subseafloor that favors CO2 reduction over NO3- reduction. Current molecular efforts will help reveal the phylogenetic identities associated with the anaerobic chemosynthetic cultures generated from subseafloor crustal fluids of the JdFR flank.