Background There are millions of deaths attributable to low levels of quality and patient safety in healthcare. One of the first steps in the journey to high reliability is to establish a solid patient safety culture. This study sought to determine the level of patient safety culture in the personnel of a specialty Hospital in Ecuador. Methods A cross-sectional study was conducted with a descriptive phase comprising the presentation of results of the patient safety culture level in the staff, and an analytical phase to determine conditioning factors of this aspect. Results 344 hospital staff answers were included in the study. The most significant group of personnel who responded to the survey corresponded to medical staff (39.83%). An overall patient safety culture level of 63.86% was determined, with higher scores observed in leadership positions (73,55%) and among the medical staff (69,90%), followed by support positions (60,97%), Other staff positions (60,44%), and nursing (56,74%). The dimensions of patient safety culture identified as strengths were teamwork and hospital management support in patient safety, and as improvement areas figured response to error, and staffing and work pace. Significant differences were found between the results of the patient safety culture domains and: staff position, working time in the hospital, working time in the current area or unit, weekly working hours, and typical contact with patients during daily activities (p < 0.05). Conclusions The patient safety culture level in the Ecuadorian context was determined, as well as its strengths and improvement areas. In this study, the support of hospital management support in patient safety stands out as a strength. Additionally, aspects of the staff were found to be significantly associated with the results of the patient safety culture dimensions indicating potential areas of focus for efforts to improve patient safety culture.