Purpose: This study was done to investigate nurses’ knowledge of, and compliance with the multidrug-resistant organism (MDRO) infection control guidelines. Methods: A survey questionnaire was developed based on the institutional and national guidelines and was administered to a convenience sample of 306 nurses in a university hospital. Results: The mean score for knowledge was 33.87 (percentage of correct answers: 82.61%). The percentages of correct answers for basic concepts, route of transmission, hand washing/protective devices and environment management were 74.27%, 94.29%, 92.90% and 75.54% respectively. The mean compliance score was 4.15 (range: 1-5). The compliance scores for education, communication, contact precaution, disinfection, surveillance culture, and hand washing were 3.29, 4.05, 4.20, 4.50, 4.40 and 4.48 respectively. Nurses indicated “lack of time (30.06%)”, “lack of means (10.78%)” and “lack of knowledge (9.48%)” as reasons for noncompliance. Conclusion: While most educational programs have focused on hand washing or use of protective devices to prevent transmission of MDRO in acute care settings, hospital nurses’ knowledge of the basic concepts of MDRO and environmental management has remained insufficient. Nurses are relatively non-compliant to the guidelines in the areas of education (staff, patient, family) and communication. Comprehensive educational programs are needed to decrease hospital infection rates and to improve the health of patients.