Previous research demonstrates that organizational culture (OC) and knowledge, attitudes, and practices of health care personnel are associated with the overall success of infection control programs; however, little attention has been given to the relationships among these factors in contributing to the success of quality improvement programs. Cross-sectional surveys assessing OC and knowledge, attitudes, and practices related to methicillin-resistant Staphylococcus aureus (MRSA) were distributed to 16 medical centers participating in a Veterans Affairs MRSA prevention initiative in 2 time periods. Factor analysis was performed on the OC survey responses, and factor scores were generated. To assess associations between OC and knowledge, attitudes, and practices of health care personnel, regression analyses were performed overall and then stratified by job type. The final analyzable sample included 2,314 surveys (43% completed by nurses, 9% by physicians, and 48% by other health care personnel). Three OC factors emerged accounting for 53% of the total variance: "Staff Engagement," "Overwhelmed/Stress-Chaos," and "Hospital Leadership." Overall, higher Staff Engagement was associated with greater knowledge scores, better hand hygiene practices, fewer reported barriers, and more positive attitudes. Higher Hospital Leadership scores were associated with better hand hygiene practices, fewer reported barriers, and more positive attitudes. Conversely, higher Overwhelmed/Stress-Chaos scores were associated with poorer reported prevention practices, more barriers, and less positive attitudes. When these associations were stratified by job type, there were significant associations between OC factors and knowledge for nurses only, between OC factors and practice items for nurses and other health care personnel, and between OC factors and the barriers and attitudes items for all job types. OC factors were not associated with knowledge and practices among physicians. Three OC factors-Staff Engagement, Overwhelmed/Stress-Chaos, and Hospital Leadership-were found to be significantly associated with individual health care personnel knowledge, attitudes, and self-reported practices regarding MRSA prevention. When developing a prevention intervention program, health care organizations should not only focus on the link between OC and the knowledge, attitudes, and practices of health care personnel, but also target programs based on health care personnel type to maximize their effectiveness.