X-ray irradiation was evaluated for improving microbial safety and the quality of flue-cured tobacco during aging. Tobacco samples were irradiated at doses of 0, 1, 2, 3 and 5kGy and stored for 12 months under normal storage conditions or in a high-humidity (RH>70%) room. Microbiological data indicated that the population of total aerobic bacteria was significantly decreased with increasing irradiation doses. In particular, a dose of 2kGy was effective for the decontamination of fungi from the tested samples, with a 0.93logCFU/g reduction for bacteria. The control and 1kGy X-ray treated tobacco samples were became rotted and moldy after the 12th month, whereas those treated with 2, 3 and 5kGy had no detectable mold during 12 months of storage at high humidity. Chemical measurements showed that irradiation up to 3kGy did not affect the total nitrogen, nicotine, reducing and total sugars, ratio of total nitrogen to nicotine and sugar-to-nicotine ratio. Furthermore, sensory evaluation results also showed that X-ray irradiation did not affect sensory scores with irradiation at a dose <3kGy. Based on these results, X-ray irradiation dose in the range of 2–3kGy is recommended for the decontamination of fungi from flue-cured tobacco.