IIonizing radiation (IR) has been broadly employed in the last decades, commonly as a diagnostic and therapeutic tool. Thus, medical staff is frequently exposed to IR during work. Persistent introduction to low doses of IR can lead to genotoxicity via stimulating the generation of reactive oxygen species. This study aimed to investigate DNA degradation and evaluate the potential impact of oxidative stress status on DNA damage in healthcare workers (HCWs) working in diagnostic radiology units. The studied groups included 21 HCWs who were occupationally exposed to IR, and 20 healthy office employees selected as unexposed control subjects who were matched with the exposed group with respect to age, sex, and inclusion criteria. Malondoaldehyde (MDA) and superoxide dismutase (SOD), as oxidant and antioxidant biomarkers, were assessed in all participants. Also, DNA degradation was quantified by a comet assay (a genotoxicity marker). The results show significant elevation in MDA, reduction in SOD, and DNA degradation in the exposed group. DNA degradation was higher with increased duration of exposure. Exposed technicians had more DNA degradation than exposed nurses and physicians. A positive correlation between comet parameters (tail length, tail moment, % tailed DNA, and DNA% in the tail) with MDA and duration of exposure was noticed. While negative correlation was demonstrated between these parameters and SOD in the exposed group. IR can lead to increased DNA damage in occupationally exposed health care workers, which is positively correlated with the duration of exposure and oxidant status.