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Background Routine assessments of patient safety culture within hospitals have been widely recommended to improve patient safety. Experts suggested that mixed-methods studies can help gain a deeper understanding of the concept. However, studies combining quantitative and qualitative approaches exploring patient safety culture are still lacking. This study aimed to explore patient safety culture as perceived by operating room professionals of two university hospitals in Sousse, Tunisia. Methods Based on a mixed-methods approach, a cross-sectional survey followed by semi-structured interviews were conducted over a period of two months (December 2019 to January 2020). This study took place in all the operating rooms of two public university hospitals in the district of Sousse, Tunisia. To collect data for this survey, the French version of the Hospital Survey On Patient Safety Culture was used. For interviews, 13 participants were selected purposively using a critical case sampling approach and a topic guide was prepared. Anonymity and confidentiality were respected. Results Overall, twelve operating rooms, with different surgical specialties, were included in the study. Survey feedback was provided by 297 professionals representing a response rate of 85.6%. Concerning patient safety culture, the 10 dimensions had low scores (below 50%) and were considered “to be improved”. The highest score was found in ‘teamwork within units’ (45%). Whereas, the lowest scores were allocated to ‘non-punitive response to error’ (22.9%), followed by “frequency of adverse event reported” (25.6%) and “communication openness” (26.3%). Per qualitative data, participants provided a more detailed picture of patient safety issues such as underreporting, absence of an effective reporting system, lack of freedom of expression, and an existing blame culture in operating rooms. Conclusions The findings of this study showed a concerning perception held by participants about the lack of a patient safety culture in their operating rooms. It seems essential to design, implement and evaluate strategies that promote a positive patient safety culture and obliterate punitive climate in operating rooms.