Electron beam to target turning X-ray (EBTTX) is an emerging irradiation technology that can potentially accelerate the breeding process of plants. The biological effects of EBTTX irradiation on the two freesia cultivars (the red freesia and the purple freesia) were investigated by establishing an irradiation-mediated mutation breeding protocol. The germination rate, survival rate, plant height, leaf number and area, root number and length of the two freesia cultivars decreased following different irradiation doses (25, 50, 75, and 100-Gy). A high irradiation dose exhibited stronger inhibition effects on these plant growth parameters, and the survival rate of the two freesia cultivars was 0.00% following the 100-Gy irradiation treatment. The median lethal dose (LD50) based on survival rates was 54.28-Gy for the red freesia and 60.11-Gy for the purple freesia. The flowering rate, flower number, and pollen vigor were significantly decreased by irradiation treatment. At 75-Gy irradiation, the flowering rate, flower number and pollen viability of the two varieties reached the minimum, exhibiting strong inhibitory effects. Meanwhile, 75-Gy irradiation significantly decreased the chlorophyll content and increased the malondialdehyde (MDA) content of the two freesia cultivars. Furthermore, as the irradiation dose increased, the changes in the micro-morphology of the leaf epidermis and pollen gradually increased according to a scanning electron microscope (SEM) analysis. These results are expected to provide useful information for the mutation breeding of different freesia cultivars and other flowering plants.