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Introduction Patient safety culture is essential for reducing the incidence of adverse events in high-risk environments, such as operating rooms. The objective of this study was to evaluate the dimensions of safety culture among healthcare personnel. Methodology A descriptive, cross-sectional, multicenter study was conducted using non-probability convenience sampling, with 372 individuals from operating rooms in four cities (three in Colombia and one in Panama) participating. Data collection complied with international (CIOMS) and national (Resolution 8430 of 1993, classified as “no risk”) ethical guidelines, and informed consent was obtained from all participants. Results: The perception of safety culture in care was rated at an intermediate level, with an average score of 8 on a scale of 1 to 10, falling short of the confidence level of 9/10. Low adherence to incident reporting was observed, with 53.8% of staff never reporting incidents or adverse events. None of the dimensions of safety culture were classified as strengths overall, as the percentage of positive responses did not exceed 60%. However, a positive relationship was found between the dimensions of Organizational Learning/Continuous Improvement and Teamwork in the Unit/Service. Conclusion: Strengthening these dimensions is key to promoting patient safety culture in the health services of these countries.