Background: DNA damage in male germ cells due to exposure to environmental and manmade physico-chemical genotoxic agents is considered as the main cause of male infertility. The aim of this study was to evaluate the effects of combined modalities (radiotherapy and chemotherapy) routinely used for cancer treatment on mouse sperm chromatin in vivo. Materials and Methods: Forty-eight mice were divided into 12 groups: 3 irradiation (1, 2, and 4 Gy), 2 drug [Actinomycin-D (ACTD) and Bleomycin (BLM)], 3 ACTD/irradiation, 3 BLM/irradiation, and a control. Mice received intratesticular injection of 7µg/25 g of Actinomycin-D and Bleomycin before irradiation with X-rays. Forty-eight hours after irradiation, mice were sacrificed and epididymis and testes were removed. Sperm DNA damage was assessed with the use of alkaline comet assay. Moreover, morphology, and motility of sperms were investigated microscopically.  Results: Result showed that drug alone had slight but not significant effect on sperm DNA damage, but significantly increased when combined with irradiation. There was a significant difference between the experimental and the control group in DNA sperm damage, but no significant differences were observed in sperm morphology (p>0.05). In the drug+4Gy group, DNA damage increased dramatically compared to the controls (p<0.01), morphology changes increased to about six times that of controls. Conclusion: Results indicate that X-ray induced DNA damage and morphological changes in sperms in a dose dependent manner. Low doses of drugs led to potentiation of radiation effect. This might be indicative of necessity for radiation protection of testes when are in field of radiotherapy.