Biological nitrogen (N₂) fixation is the main input of fixed nitrogen to ecosystems on Earth. Nitrogen isotope fractionation during this process is a key parameter for understanding the nitrogen cycle, however, relatively little is known about its regulatory mechanisms. Here we examine the effects of varying CO₂ concentrations on biomass &delta;¹⁵N signatures of the cyanobacterial diazotrophs <em>Trichodesmium</em> <em>erythraeum </em>and<em> Crocosphaera watsonii</em>. We show that these organisms produce biomass up to ~3 &permil; lower in &delta;¹⁵N under either decreased (~180 &micro;atm) or elevated (~1400 &micro;atm) CO₂concentrations in comparison to modern levels (~380 &micro;atm). Our results pointed towards changes in nitrogenase enzyme efficiency in response to CO₂ perturbations impacting isotopic fractionation during N₂ fixation and thus the biomass &delta;¹⁵N. This study contributes to an improved interpretation of the observed fluctuations in the &delta;¹⁵N records, and thus the past nitrogen cycle on Earth.