Sunflower plants (Helianthus annuus L.) in a CO2-enriched atmosphere (eCO2) were used herein to examine the developmental and physiological effects of biofertilization with mycorrhizae (Rhizophagus irregularis). The eCO2 environment stimulated colonization using R. irregularis mycorrhizal fungi, as compared to plants grown under ambient CO2 conditions (aCO2). This colonization promotes plant growth due to an increased nutrient content (P, K, Mg, and B), which favors a greater synthesis of photosynthetic pigments. Biofertilized plants (M) under eCO2 conditions have a higher concentration of carbon compounds in their leaves, as compared to non-biofertilized eCO2 plants (NM). The biofertilization (M) of sunflowers with R. irregularis decreased the C/N ratio, as compared to the NM plants, decreasing the hydrogen peroxide content and increasing the antioxidant enzyme activity (catalase and APX). These results suggest that sunflower symbiosis with R. irregularis improves the absorption of N, while also decreasing the plant’s oxidative stress. It may be concluded that biofertilization with mycorrhizae (R. irregularis) may potentially replace the chemical fertilization of sunflower plants (H. annuus L.), resulting in more environmentally friendly agricultural practices. This information is essential to our understanding of the mechanisms influencing the C and N dynamic in future climate change scenarios, in which high CO2 levels are expected.