Introduction Patient safety is an issue of global concern; however, health-care organizations have lately observed to pay more attention to the importance of establishing a culture of safety. The study aimed to assess the level of patient safety culture and associated factors among health-care providers at the University of Gondar comprehensive specialized hospital, Northwest Ethiopia, 2020. Methods A cross-sectional study design supported by the qualitative approach was conducted from March 15 to May 15/2020. A stratified simple sampling technique was used to select 575 study participants. The standardized tool, which measures 12 safety culture dimensions, was used for data collection. Bivariate and multivariable linear regression analyses performed using SPSS version 23. The significance level was obtained at 95% CI and p-value <0.05. For the qualitative part, a semi-structured interview guide with probing was used. Data were analyzed thematically using open code software version 4.02. Results The overall level of positive patient safety culture was 45.3% (95% CI: 44.7, 45.9) with a response rate of 92.2%. Factor analysis indicated that female, masters, participation in patient safety program, adverse event report, hospital management encourage reporting event and resource were positively associated with the patient safety culture. Whereas divorced/widowed, midwives, anesthetist, medicine, pediatrics, emergency, outpatient, pharmacy, direct contact with patients, and hospital management blame when medical errors happened were negatively associated. The in-depth interview revealed that teamwork, health-care professionals’ attitude toward patient safety and patient involvement as important factors that influence patient safety culture. Conclusions and Recommendations The overall level of positive patient safety culture was low. All variables except age, training, working hour, and working experience were factors significantly associated with the patient safety culture. Health-care policy-makers and managers should consider patient safety culture a top priority, and also create a blame-free environment that promotes event reporting.