Objective The aim of the study was to identify and rank leading healthcare quality and patient safety challenges of general hospitals in Spain. Methods A novel online cross-sectional survey for hospitals with 150 or more beds carried out between June and September 2018. Sample frame is hospitals of the National Catalogue of Hospitals of the Ministry of Health in Spain (N = 888). Eligibility criteria are quality experts of clinical quality and/or patient safety services of general hospitals with 150 or more beds. Challenges were ranked using a risk priority number (RPN) calculated from the product of severity, frequency, and detectability scores. Results Targeted hospitals were 234. The contact rate was 97.4%, representing 63% of total beds nationwide. One hundred hospitals completed the questionnaire. According to the RPN, the top five challenges were incorrect hand hygiene of health professionals (mean RPN = 334.5, SD = 198.5), ineffective interprofessional communication (mean RPN = 334.3, SD = 169.5), medication errors in transitions of care (mean RPN = 254.0, SD = 151.0), low reporting rates of patient safety incidents and adverse events (mean RPN = 252.3, SD = 176.3), and antimicrobial resistance due to inappropriate use of antibiotics (mean RPN = 243.5, SD = 158.7). Conclusions This pioneer study of ranking quality and patient safety challenges of hospitals in Spain provides an evidence-based and context-specific foundation for quality improvement decision-making.