Brassica oleracea is one of the important vegetable crops in China. Its seeds are primarily imported. Hence, developing innovative varieties belonging to China is an urgent requirement. Since broccoli is a cross-pollinated crop systematic selection and cross-breeding are challenging, and the application of radiation mutagenesis breeding in broccoli has not been reported. In this study, the seeds of Brassica oleracea were irradiated using carbon ion beams to investigate the biological effects of these beams on broccoli. The growth indices, antioxidant enzyme activity, photosynthetic indices, and chlorophyll fluorescence were detected at the seedling stage. The results revealed that irradiation with 100–500 Gy exhibited no significant effect on the germination of Brassica oleracea, while the germination of seeds was significantly inhibited at 600 Gy. After irradiation with 100–600 Gy, the root length, shoot length, seedling height, and leaf area decreased with the increase in irradiation dose. The Median lethal dose (LD₅₀) of Brassica oleracea irradiated using a carbon ion beam was 415.89 Gy, and the dose that halved the root length was 495.12 Gy. After irradiation, the activities of superoxide dismutase (SOD) and peroxidase (POD) were higher than that of the control plants, while the activity of catalase (CAT) was lower than that of the control plants. The content of malondialdehyde (MDA) increased significantly at a dose of 400 Gy. The photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) showed an increasing trend initially, and then a decreasing trend with an increasing irradiation dose, and the highest value was detected at a dose of 300 Gy. Net photosynthesis, transpiration rate, and stomatal conductance exhibited a negative correlation with irradiation dose, while the non-photosynthetic quenching coefficient exhibited a significant increase after irradiation. The findings indicated that heavy ion beam radiation inhibited the growth of broccoli plants and affected the activity of antioxidant enzymes and photosynthesis. The study provided basic data for the radiation mutation breeding of Brassica oleracea.