Patient safety culture is crucial for every health care institution, as a lack of it may harm patients seeking treatment. The current study aimed to identify the level of safety culture and assess the knowledge, attitude, and perception of patient safety culture among healthcare providers (HCPs') in tertiary hospital settings. A cross-sectional study was conducted among HCPs from two private tertiary hospitals in Johor and Selangor. A structured validated questionnaire, including the Hospital Survey on Patient Safety Culture, was used to assess the level of patient safety culture in these hospitals. In this study, the calculated sample size was 320, and all 550 eligible participants from both hospitals were approached to participate. However, only 158 responded, resulting in a response rate of 49.38%. The majority of the HCPs (n = 110; 69%) rated their hospital as very good or excellent in maintaining an overall patient safety culture. The study revealed that communication about the errors (PRR = 80) and organizational learning and continuous improvement (PRR = 74) were good in their hospital settings. However, staffing and work pace (PRR = 28), response to errors (PRR = 40), reporting patient safety events (PRR = 48), and handoffs and information exchange (PRR = 39) were inadequate. These findings indicate the negative attitudes among HCPs and the need for further improvement to maintain a culture of patient safety. HCPs in the study settings had optimal knowledge but negative attitudes towards the culture of patient safety in their organization. Inadequate staffing, work pace, and a lack of response to mistakes were commonly observed, which may increase the chances of errors and pose health threats to patients that need to be addressed immediately. Every healthcare organization is urged to address the issue of patient safety culture as a matter of urgency.