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 (N-2) and carbon (C) fixation rates of these colonies were compared over CO2 conditions ranging from similar to 18 Pa (equivalent to last glacial maximum atmospheric P-CO2) to similar to 160 Pa (predicted for similar to year 2200). Our results indicate that elevated P-CO2 has no consistent significant effect on rates of N-2 or C fixation by Trichodesmium colonies in the NPSG under present environmental conditions. Differences between P-CO2 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 N-2-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 P-CO2 in previous laboratory experiments. Our handpicked Trichodesmium colonies included a substantial number of organisms other than Trichodesmium with the metabolic capacity for N-2 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 N-2 or C fixation rates, because different species are known to have different specific affinities for CO2.