Understanding the biological effects of neutron mixed-beam irradiation used for boron neutron capture therapy (BNCT) is important in order to improve the efficacy of the therapy and to reduce side effects. In the present study, cell viability and DNA double-strand breaks (DNA-DSBs) were examined in Chinese hamster ovary cells (CHO-K1) and their radiosensitive mutant cells (xrs5, Ku80-deficient), following neutron mixed-beam irradiation for BNCT. Cell viability was significantly impaired in the neutron irradiation groups compared to the reference gamma-ray irradiation group. The relative biological effectiveness for 10% cell survival was 3.3 and 1.2 for CHO-K1 and xrs5 cells, respectively. There were a similar number of 53BP1 foci, indicators of DNA-DSBs, in the neutron mixed-beam and the gamma-ray groups. In addition, the size of the foci did not differ between groups. However, neutron mixed-beam irradiation resulted in foci with different spatial distributions. The foci were more proximal to each other in the neutron mixed-beam groups than the gamma-ray irradiation groups. These findings suggest that neutron beams may induce another type of DNA damage, such as clustered DNA-DSBs, as has been indicated for other high-LET irradiation.