Background: Handling of cytotoxic drugs always raises concerns particularly in preparation and administration of them. Health care workers have been handling these agents in the open areas of wards in the absence of infection control measures. Objective: The aim of this study is to evaluate the handling of cytotoxic drugs in pediatric Hematology/Oncology Unit at Tanta University Hospitals and potential exposure risk to cytotoxic drugs and in addition to evaluate hand flora of health care workers (HCWs). Subjects and methods: A performance checklist was used to assess the practices of nurses and workers; in addition, hand print was also used to assess hand flora of HCWs on blood and MacConkey agar plate (4clinical pharmacists, 11nurses and 6workers) were included.Results: All nurses (100%) did not follow guidelines regarding (patient care, preparation, administration, spill, waste disposal, risk behaviors and excreta management). Concerning risk exposure assessment, only (9.1%) suffered from nausea of unknown cause, (27.3%)fromURTIs symptoms and (18.2%)from headache. Decontamination efficacy was evaluated by hand print technique and according to the total bacterial load detected, it was found that 54.5%, 25% and 0% of nurses, clinical pharmacists and workers respectively had excellent performance regarding hand hygiene. Moreover 50%, 25% and 9.1% of workers, clinical pharmacists and nurses had very bad performance of hand hygiene respectively. Conclusion:  The study clarifies the need of continuous education on handling of cytotoxic drugs (CDs), give more attention to a truth that the cytotoxic agent can be critical to them.