Background The development of a culture of safety can bring about an improvement in patient safety and quality of care. We aimed to investigate patient safety culture within an acute-care hospital setting in Greece. Methods A cross-sectional quantitative study was conducted using the Hospital Survey on Patient Safety Culture (HSOPSC) questionnaire in two large acute-care hospitals in Greece. We examined questionnaire’s factorial structure using confirmatory and exploratory factor analysis. We assessed scales’ internal consistency, test–retest reliability and construct validity. To assess changes in patient safety culture, we conducted surveys at two points in time (2009 and 2014). Regression analysis examined whether any changes in scale scores could have been due to quality programs implementation or participants’ professional characteristics. Results Six scales with satisfactory psychometric properties emerged, namely ‘Teamwork Across Hospital Units’, ‘Teamwork Within Hospital Units’, ‘Feedback and Communication About Error’, ‘Frequency of Event Reporting’, ‘Supervisor/Manager Expectations and ‘Actions Promoting Safety’, ‘Hospital Management Support for Patient Safety’. Overall ratings of patient safety did not change over time. However, one of the two institutions experienced an increase in reported events. Conclusions The HSOPSC is a valuable tool for assessing patient safety culture in Greece. Moreover, despite the radical reduction in the financing of the Greek National Health System reported between 2009 and 2014, patient safety culture did not deteriorate. A worldwide trend towards increasing patient safety awareness, along with the increasing effort of hospitals’ administration to support patient safety might explain this lack of differences between the two points in time.