The expansion of anti-cancer chemotherapy has led to a significant concern about the health and safety work conditions of the personnel handling these drugs that have been characterized by the International Agency for Research on Cancer as hazardous to human. However, no reliable monitoring test of occupational exposure is currently clinically applied for the medical surveillance of these employees. Based on a descriptive self-evaluation survey, we have previously shown inadequate health and safety design and practices related to chemotherapeutic drug handling in Greek hospitals. In the present study, we use a quantitative and objective genotoxicity method, the comet assay, to evaluate occupational exposure to chemotherapeutics. DNA single strand breaks as revealed by comet in peripheral mononuclear blood cells were significantly higher in a group of employees involved in the preparation, handling or administration of cancer chemotherapeutic agents that in a control group. Comet data were correlated to demographic/epidemiological health data of the subjects as well as to the self-evaluation subjective occupational exposure data. Our results demonstrate clearly genotoxic effects related to occupational exposure to chemotherapeutics, indicating insufficient safety design and practices. They also suggest that SSBs could be an appropriate biomarker for evaluating genotoxicity in these employees. The comet assay in peripheral blood could provide an easy tool of the Health and Safety Department in hospitals for the exposure monitoring.