Background Antineoplastic drug exposure is a major problem in regard to caregivers’ health. The aim of the present study was to assess the perception, knowledge, and handling practices of all occupation level categories of two oncology day hospitalization units and two compounding units regarding the risk of exposure to antineoplastic drugs. Methods This descriptive study, performed through face-to-face interviews, concurrently assessed the perception, knowledge, and handling practices of antineoplastic drugs in five different job categories in four different settings. This work was part of a larger comprehensive project examining surface and blood contamination. Different scores were assigned to evaluate responses to a questionnaire about the perception, knowledge, and handling practices of healthcare workers, a risk global score including a risk perception score, and education/knowledge and handling practices scores. Results In the survey, continuous training was associated with the global risk score (p = 0.03), particularly with the handling practices risk score (p = 0.01). Job category was also significantly associated with the global risk score (p < 0.001), particularly with the handling practices risk score (p < 0.001) and the education/knowledge score (p < 0.001). Pharmacy technicians had the highest score regarding risk perception (71.4%), indicating a higher perception of risk, and had a lower score regarding handling practices (25.0%) as well as a lower score (15.7%) regarding risk knowledge. Nurses and physicians had a high score (50%) regarding the risk of handling practices and a score of 57.1% regarding risk perception, indicating an increased perception of safety. Auxiliary caregivers had the highest global score (43.5%) and a score of 30.0% regarding handling practices. Conclusions This study identified significant differences among healthcare workers depending on job categories in the antineoplastic drug handling practices and in the knowledge of the risks associated with occupational exposure to antineoplastic drugs. These differences were particularly important between trained and untrained participants, revealing the importance of implementing a continuous training program.