Reprocessing of reusable medical devices (RMD) play an important role in the prevention of health-care associated infections. In this perspective, this study aimed to evaluate the impact of a training program intervention on health-care workers' (HCWs) knowledge and hospital practices about the reprocessing of RMD as a main strategic axis of infection control. We led a pre-post quasi-experimental study to evaluate knowledge of HCWs and to assess hospital practices in reprocessing RMD before and after a training intervention from May to November 2020 in Hedi Chaker University Hospital. A self-administrated questionnaire was distributed for each participant to assess knowledge. To evaluate practical skills, an audit grid was used. Overall, 30 participants were included. All HCWs had a fair level of knowledge before training. The knowledge score (KS) has significantly risen from pre to post-training (64/100 vs. 80/100; P<0.001). When stratified by RMD type, a significant increase was noted on post intervention in KS about thermo-sensitive RMD reprocessing (P=0.044) and sterilization of thermo-resistant RMD (P=0.004). As for practice assessment, 5 (62.5%) departments had a fair level of practice. The conformity score (CS) did not significantly change from baseline to post-follow-up (53.5 vs. 56.2; P=0.06). According to the audit sections, we did not find a significant change in the pre- and post-intervention CS in sterilization process (70 vs. 80; P=0.06) and in the cleaning and decontamination steps (66.7 vs. 67; P=0.18). As opposed to practical skills, positive impact of the training intervention on HCWs knowledge was noted, which underlined the gap between theory and practice.