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Abstract Background The patient safety culture survey is an essential tool for assessing beliefs, values, and behaviors related to patient safety at different levels of a healthcare organization. The main objective is to identify areas for strengthening and improvement opportunities, aiming to promote a safe environment for patients. The survey was conducted by a large Brazilian diagnostic service, using the Medical Office Survey on Patient Safety Culture, developed by the Agency for Healthcare Research and Quality (AHRQ), as the tool. The dimensions assessed, such as teamwork, open communication, organizational learning, and management support, directly influence the quality of care provided. Methods The 2nd edition of the survey was conducted in several stages, including preparation, dissemination, data collection, analysis, and communication of results. Data collection took place between October 2024 and November 2024, with a focus on involving 21,153 eligible professionals. A total of 3,949 responses were received, of which 3,537 were eligible, resulting in a response rate of 17%. When compared to the literature (Sckolnick, 2021) and the 1st edition of the survey conducted in 2022, the expected percentage to make the result representative was a minimum participation of 10% of the eligible audience. Results The survey results indicated that 63% of respondents rated the safety culture as positive, while 21% considered it neutral and 16% negative. The dimensions with the highest percentage of positive responses were Organizational Learning/Continuous Improvement (78%), Teamwork Between Units (75%), Feedback and Communication of Errors (73%), Frequency of Adverse Event Reporting (73%), and Teamwork Within Units (58%). However, some dimensions showed opportunities for improvement, such as Non-Punitive Response to Errors (30% positive responses) and Adequacy of Staffing (41%). It was also observed that 52% of respondents reported never having made a patient safety incident report. The overall perception of safety was considered excellent or very good by 63% of respondents at the institutional level and 60% at the regional level. Compared to the 1st edition conducted in 2022, there was only a 2% difference, with just one dimension showing a decrease in percentage: Teamwork Within Units, which dropped from 76% in 2022 to 58% in 2024. Conclusion The survey highlighted that the safety culture is strong in several dimensions, such as communication and teamwork, but there are still opportunities for improvement, especially regarding non-punitive responses to errors and reporting of safety events. Implementing corrective and preventive actions, with a focus on continuous learning and management support, will be crucial to further strengthen the patient safety culture. Communicating the results and engaging all levels of the organization will be essential to promote a safer and more effective work environment, benefiting both professionals and patients. To overcome these barriers, it is essential to invest in strategies that prioritize the continuous training of professionals, the creation of evidence-based protocols, and the strengthening of leadership committed to patient safety. Adopting practices that encourage an environment of trust and learning, where errors are seen as opportunities for improvement, is also fundamental to consolidating the safety culture.