Background and Objective: Infectious Health Care Waste (HCW) could induce nosocomial infections, putting the health of medical staff and patients at risk. Proper HCW management should therefore be strictly followed as part of a comprehensive and systematic approach to hospital hygiene and infection control. We aimed at evaluating HCW management among health workers on both sides: knowledge and practice. Methods: We performed a cross sectional study on September 2021 at all departments of the University Hospital Mahdia (Tunisia). We used an evaluation grid to assess the accessibility of resources and the property of practices. A self-administered questionnaire, validated by the Regional Office for Nosocomial Infection Control in France was used to assess HCW management knowledge. Results: A total of 160 health workers responded to the on-site separation questionnaire with a mean age of 32.3±7.9. Most of them were practicing at medical departments (56.9%). For ordinary waste, 94.4% of professionals knew how to manage source segregation. Professionals with less than 20 years of experience had significantly better knowledge of on-site sorting of ordinary HCW (96.6% vs 66.7%, p=0.002). Regarding septic waste, 84.4% of health workers had good level of knowledge. After binary logistic regression we found that doctors had lower level of knowledge about the on-site sorting comparing to paramedical staff (34.1% vs 65.9%, OR=0.4, 95%CI=0.171-0.966). To evaluate resources and practices, we observed 59 sites at 24 units. Over all compliance rate of resource’s accessibilities was 88.1%. Sharp containers were unavailable in 39% of sites. However, compliance rate of good practice was 6.8%. In 44.1% of observations health providers placed infectious waste in general HCW containers. Conclusions: Our study revealed a gap between knowledge and practices of HCW among health care workers. A comprehensive waste management plan is needed for safe and efficient waste management.