We conducted 11 independent short-term carbon dioxide (CO2) manipulation experiments using colonies of the filamentous cyanobacteria Trichodesmium isolated on three cruises in the North Pacific Subtropical Gyre (NPSG). Dinitrogen (N2) and carbon (C) fixation rates of these colonies were compared over CO2 conditions ranging from ~ 18 Pa (equivalent to last glacial maximum atmospheric PCO2) to ~ 160 Pa (predicted for ~ year 2200). Our results indicate that elevated PCO2 has no consistent significant effect on rates of N2 or C fixation by Trichodesmium colonies in the NPSG under present environmental conditions. Differences between PCO2 treatments were not modulated by phosphorus amendments, iron amendments, or light level. Sequencing the hetR, nifH, 16S, and internal transcribed spacer genes of Trichodesmium colonies revealed a highly diverse community of Trichodesmium and other N2-fixing colony-associated organisms. The species composition of Trichodesmium demonstrated spatiotemporal variability, but over half of total sequences were phylogenetically closely related (> 99% hetR sequence similarity) to isolate H9-4 of T. erythraeum, which showed no response to elevated PCO2 in previous laboratory experiments. Our handpicked Trichodesmium colonies included a substantial number of organisms other than Trichodesmium with the metabolic capacity for N2 and C fixation. We suggest that the diverse assemblage of Trichodesmium species and coexisting microorganisms within the colonies can explain the lack of an observed CO2 enhancement of N2 or C fixation rates, because different species are known to have different specific affinities for CO2.