Background Patient safety (PS) is a fundamental component of healthcare quality. Patient Safety Culture (PSC) assessment provides an organization with insight of perceptions and attitudes of its staff related to patient safety. In addition, it is meant to improve performance rather than blaming individuals. This study aimed to assess patient safety culture from the health care staff perspective in El-Shatby University Hospital for Gynecology and Obstetrics. Methods A descriptive cross-sectional study was conducted. The study was conducted at El-Shatby University Hospital for Gynecology and Obstetrics from November 2020 to January 2021. The target participants were assistant lecturers, residents, and head nurses in charge during the field study period. The number of potential participants who fulfilled the inclusion criteria (in charge during the period of data collection and working in the hospital for more than 3 months) was 83; the twelve participants who participated in the pilot study were excluded. The total number of participants who agreed to participate in the study was 66 participants (38 residents, 18 assistant lecturers, and 10 head nurses) out of 71 potential participants representing a 92.9% response rate. A structured self-administered questionnaire format adapted from Hospital Survey on Patient Safety Culture (HSOPSC) questionnaire was distributed anonymously to the participants. The questionnaire has 42 items measuring twelve patient safety culture dimensions: teamwork within the unit, supervisors’ expectations and actions to promote patient safety, feedback and communication about error, organizational learning, communication openness, overall perception of patient safety, hands-off and transitions, teamwork across units, frequency of events reported, management support for patient safety, staffing, and management support for patient safety. Except for two items that are responded on a five-point frequency scale (never, rarely, sometimes, most of the time, and always) the majority of patient safety culture questions are answered on a five-point agreement scale (strongly disagree, disagree, neutral, agree, and strongly agree), with a higher score indicating a more favorable attitude toward patient safety. Results The overall average positive percent score was 45.4%. Average positive response percentages to individual items ranged from 28.8 to 81.8%. No domain had an average positive percent score of more than 75%. Out of the twelve dimensions of patient safety culture included in the HSOPSC questionnaire, “the teamwork within unit” domain had the highest average positive percent score (62.1%) among all participants. On the other hand, the “Non-punitive response to error” domain had the lowest score (18.9%). More than half (57.6%) of the participants rated patient’s safety at the hospital as acceptable. Conclusion Investing in practices that strengthen patient safety is crucial if the hospital is to improve overall performance and quality of services. The present study displays a frail patient safety culture (PSC) in the majority of the domains. All the domains should be considered of high priority focused areas for remark and reformative tasks. Continuous training programs of the staff on patient safety to improve their perception of safety culture are necessary. All PSC composites need improvement starting with regular assessment of PSC along with continuous monitoring and increasing the healthcare providers’ awareness of demanded PSC.