Introduction: Operating room safety is a major imperative. The surgical safety checklist (CL), developed by the World Health Organization (WHO), is an essential tool to reduce surgical risks and improve patient safety. Objective: This study aimed to assess the level of knowledge and use of the checklist among healthcare professionals in operating rooms in Senegal. Methods: This was a multicenter cross-sectional study conducted over three months, from September to December 2024. Data were collected using an anonymous questionnaire, available in both paper and digital formats. Data analysis was performed with SPSS v.29 and Excel 2021. Results: The study included 110 professionals, with 56% based in Dakar and 44% in other regions. Participants were mainly surgeons (45%), anesthesiologists (35%), and paramedical staff (25%), with an average professional experience of 6.7 years. Although 94% of professionals were aware of the checklist, only 36% used it systematically. The main barriers identified were lack of training (53%), workload (31%), and resistance related to work habits (16%). Furthermore, 92.7% of participants emphasized that training initiatives (workshops, online sessions, or a combination) could facilitate adoption and implementation of the checklist. Finally, 98.5% of participants perceived a positive impact of the checklist on patient safety. Conclusion: The checklist is an essential tool to improve patient safety in the operating room. Its integration must be part of a broader quality improvement strategy. However, adoption in Senegal is hindered by lack of training, organizational constraints, and entrenched work habits. To optimize its use, it is crucial to strengthen training, improve working conditions, and promote a systemic approach centered on safety.