Antineoplastic drugs used in the treatment of cancers have an intrinsic toxicity, because of their genotoxic, teratogenic and carcinogenic properties. Their use is recognized as an occupational hazard for healthcare workers (HCWs) who may be exposed. The aim of this article is to present biological and environmental monitoring data collected in twelve French hospitals over eight years. Urine samples were collected from a wide range of HCWs (250 participants) from pharmacy and oncology units, including physicians, pharmacists, pharmacy technicians, nurses, auxiliary nurses and cleaning agents. The investigated drugs were cyclophosphamide, ifosfamide, methotrexate and 5-fluorouracil. Wipe samples were collected from various locations in pharmacy and oncology units. More than 50% of participants, from all exposure groups, were contaminated to either drug, depending on the unit, the day or the task performed. However, workers from oncology units were more frequently exposed than workers from pharmacy units. Significant contaminations were observed in various surfaces in pharmacy and oncology units, highlighting potential sources of exposure. Risk management measures should be implemented to reduce and maintain exposures at lowest possible levels. In addition, regular exposure assessment, including biological and environmental monitoring, is recommended to ensure the long-term efficiency of the prevention measures.