Bacteriogenic iron oxides in natural environments are characterized by an abundance of ferrihydrite precipitates intermixed with bacterial structures that commonly resemble those produced by the lithoautotrophic microorganisms Gallionella ferruginea and Leptohtrix ochracea. These species have been inferred to play a causal role in the formation of bacteriogenic iron oxides, providing a pathway for the reduction of CO2 and the depletion of 13C in the organic constituents of bacteriogenic iron oxides. In this study, stable carbon isotope fractionation was determined for bacteriogenic iron oxide samples collected from submarine hydrothermal vents (Axial Volcano, Juan de Fuca Ridge), subterranean (Äspö Hard Rock Laboratory, Sweden) and surficial (Chalk River, Canada) groundwater seeps, and cultures of G. ferruginea. Data were also collected from ferrihydrite samples lacking evidence of bacteria from Bounty Seamount in the vicinity of Pitcairn Island. The mean δ13C (‰) of ferrihydrite was determined to be −15.87‰ ± 4.96‰ for the samples from Axial Volcano, −24.97‰ ± 0.43‰ for Äspö, −27.80‰ ± 0.85‰ for Chalk River, −29.3‰ ± 0.2‰ for the microbial culture, and −8.43‰ ± 1.89‰ for the samples from Pitcairn. Samples with the highest concentration of organic carbon also had the lightest δ13C in a logarithmic relationship. The consistency of carbon isotope values in relation to the presence of iron-oxidizing bacteria from natural and laboratory samples is interpreted as the ability of these microorganisms to fractionate carbon. The potential of this fractionation to serve as a biosignature holds promise when the resistance of carbon and bacteriogenic ferrihydrite to diagenesis is taken into consideration.