Mycotoxins are toxic secondary metabolites produced by fungal pathogens that infect cereal crops such as wheat and barley. Crop susceptibility to fungal infection and mycotoxin contamination is dependent on the environment, and climate change is predicted to increase the frequency and severity of conditions that favor contamination. However, it is unclear how rising atmospheric carbon dioxide (CO2) will impact mycotoxin production. We hypothesized that atmospheric CO2 levels would influence mycotoxin production. To test this hypothesis, we inoculated grain of two barley cultivars with three diverse strains of Fusarium graminearum, a devastating mycotoxigenic pathogen of cereals. The inoculated grain was placed into growth chambers controlled at 400 ppm (ambient) or 1000 ppm (elevated) CO2 conditions. The fungus was allowed to grow, colonize the grain, and produce mycotoxins for 14 days. The grain was then collected, lyophilized, pulverized, and separated for molecular and metabolic analyses. Fungal biomass was estimated by quantifying DNA using Fusarium species-specific primers. Mycotoxin profiles were determined using gas chromatography mass spectroscopy and liquid chromatography mass spectroscopy. Results suggest that F. graminearum mycotoxin production was influenced by elevated CO2 in a manner that was dependent on barley cultivar and F. graminearum strain. This study provides valuable information needed to assess future food safety risk and will be of interest to farmers and regulatory agencies.